Isolation of Nascent Transcripts with Click Chemistry
UNIT 4.24
Ozlem Yildirim1,2 1
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 2 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Steady-state levels of cellular RNA are determined by both transcriptional rate and RNA half-life. Commonly used methods for transcriptional analysis are only capable of profiling total RNA and do not distinguish changes in synthesis and decay rates. Hence, a better understanding of the temporal dynamics of cellular response for a given condition at the transcriptional level requires techniques for the analysis of nascent transcripts. Here we describe a protocol that allows isolation of nascent transcripts with a copper-catalyzed azide-alkyne C 2015 by cycloaddition (CuAAC) also known as a click chemistry reaction. John Wiley & Sons, Inc. Keywords: nascent RNA r metabolic labeling r click chemistry
How to cite this article: Yildirim, O. 2015. Isolation of nascent transcripts with click chemistry. Curr. Protoc. Mol. Biol. 111:4.24.1-4.24.13. doi: 10.1002/0471142727.mb0424s111
INTRODUCTION To better understand the cellular response to a stimulus or a condition it is important to determine the acute changes in gene expression programs upon that stimulus. This requires a method that can discriminate the nascent transcripts from total cellular RNA, as differences in transcript half-lives of different genes greatly obscure the true transcriptional response. In addition to its use for studying temporal transcriptional activity, such a protocol can be used for analysis of RNA decay rates. Recently, different approaches, which mainly utilize the metabolic tagging of nascent RNAs with nucleoside analogs, have emerged (Core et al., 2008; Dolken et al., 2008; Rabani et al., 2011; Curanovic et al., 2013). Similarly, the protocol in this unit utilizes metabolic incorporation of nucleoside analog 5-ethynyl uridine (EU) into newly synthesized RNAs. It has been shown in vivo that EU incorporation is specific to RNA molecules and it does not nonspecifically label cellular DNA (Jao and Salic, 2008). Then azide-containing biotin molecules are covalently attached to the alkyne-modified uridine-containing transcripts with the assistance of copper catalysis in vitro (Paredes and Das, 2010). Biotinylated EU residues in the nascent transcripts allow the efficient isolation with streptavidin-coated magnetic beads. The Basic Protocol describes labeling and isolation of newly synthesized RNAs from synchronized cells. These RNAs can be sequenced to determine the spectrum of RNAs that are synthesized. It is frequently useful to determine the efficiency of incorporation of the EU label prior to performing further analysis of the sample. The Support Protocol describes how to accomplish this procedure.
Preparation and Analysis of RNA Current Protocols in Molecular Biology 4.24.1-4.24.13, July 2015 Published online July 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142727.mb0424s111 C 2015 John Wiley & Sons, Inc. Copyright
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BASIC PROTOCOL
ISOLATION OF CELL-CYCLE-SPECIFIC NASCENT TRANSCRIPTS WITH CLICK CHEMISTRY This protocol allows isolation of newly synthesized RNAs at any stage of the cell cycle. First, cells are synchronized at the G1/S border. After release into the S phase, the cell culture medium is changed to EU-containing medium for 1 or 2 hr to label nascent RNA. Labeled RNA is then isolated by biotin-streptavidin interaction (Fig. 4.24.1).
Materials
Isolation of Nascent Transcripts
Cultured cells (e.g., hTERT immortalized RPE1 cells; 2 × 106 to 5 × 106 cells for each experiment) RPE1 medium (see recipe) 5-Ethynyl uridine (Berry & Associates, cat. no. PY7563) TRIzol reagent (Life Technologies, cat. no. 15596-026) Nuclease-free water (Ambion, cat. no. AM9932) Chloroform (Sigma-Aldrich, cat. no. C2432) 100% and 80% ethanol Isopropanol (Sigma-Aldrich, cat. no. I9516) 10× Baseline-ZERO reaction buffer Baseline-ZERO DNase (Epicentre, cat. no. DB0715K) RNA Clean & Concentrator kit (Zymo Research, cat. no. R1015) Acetonitrile (Sigma-Aldrich, cat. no. 34998) 1 M Tris·HCl, pH 7.5 Sodium-L-ascorbate (Sigma-Aldrich) Copper sulfate (CuSO4 ; Sigma-Aldrich, cat. no. 451657) Biotin-dPEG 7-azide (Quanta BioDesign, cat. no. 10825) N,N,N ,N ,N -pentamethyldiethylenetriamine (PMDTA; Sigma-Aldrich, cat. no. 369497) L-Mimosine (Sigma-Aldrich, cat. no. M0253) Glycogen (Ambion, cat. no. AM9510) Dynabeads MyOne Streptavidin C1 magnetic beads (Life Technologies, cat. no. 65001) 3 M sodium acetate, pH 5.5 (Ambion, cat. no. AM9740) RNasin Plus Rnase inhibitor (Promega) Ribo-Zero Gold Magnetic kit (Epicentre, cat. no. MRZG12324) SuperScript VILO cDNA synthesis kit (Life Technologies, cat. no. 11754-050) RNAse-free water 0.5 M EDTA 1 N NaOH 1 M HEPES 100 mM DTT (Life Technologies, cat. no. 18080-044) SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080-044) Random primers (Life Technologies, cat. no. 48190011) 10 mM dNTPs First Strand buffer (Life Technologies, cat. no. 18080-044) 5× Second Strand buffer (Life Technologies, cat. no. 10812-014) E. coli DNA ligase (New England BioLabs, cat. no. M0205S) E. coli DNA polymerase (New England BioLabs, cat. no. M0209L) E. coli RNase H (New England BioLabs, cat. no. M0297S) Uracil DNA glycosylase (UDG; New England BioLabs, cat. no. M0280S) TNE bead-blocking buffer (see recipe) TNE2.0 (see recipe) TNE0.2 (see recipe) Wash buffer 65 (see recipe)
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Figure 4.24.1 (A) Schematic of experimental design of the in vivo assay. (B) Nascent transcripts are labeled with EU at different time points into S phase. RNA is isolated and conjugated to UV-cleavable biotin-azide via a click reaction. As expected, S phase-specific histone H2B was enriched in EU-labeled samples but not in unlabeled samples. (C) Xist RNA which is known to exist throughout the cell cycle is enriched in EU-labeled samples but not in unlabeled samples. Preparation and Analysis of RNA
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RNaseZap (Ambion, cat. no. AM9780) Amicon YM-10 Ultra-0.5 Centrifugal Filter Unit (EMD Millipore, cat. no. UFC5010BK) Ampure XP beads (Beckman Coulter Genomics, cat. no. A63881) Dyna Mag magnetic stand (Life Technologies, cat. no. 12321D) Cell lifter (Corning, cat. no. 3008) Low-binding, nuclease-free 1.7-ml tubes (Eppendorf, cat. no. 022431021) Thermomixer (Eppendorf, cat. no. 022670107) Rotator (Thomas Scientific, cat. no. 1217H25) Thermocycler NOTE: All solutions must be prepared with Rnase-free water.
Synchronization and metabolic labeling of cultured cells with EU 1. Grow hTERT immortalized RPE1 cells to 75% to 80% confluence. Trypsinize and plate 3 × 106 cells per 15-cm dish in RPE1 medium with a final concentration of 450 μM mimosine to arrest cells at G1/S border. This protocol can work with other cell types once the cell type-specific synchronization conditions are determined. We chose hTERT-RPE1 cells for two main reasons: first, they are diploid immortalized cells; second, they can be very efficiently synchronized at the G1/S border of the cell cycle.
2. After 21 hr of mimosine synchronization, wash cells twice with 15 ml PBS and release them into S phase in regular culture medium. For hTERT-RPE1 cells it takes approximately 3 hr for 20% of the population to enter S phase. We recommend testing different timing and conditions followed by fluorescenceactivated cell sorting (FACS) to determine the best conditions for desired synchronization.
3. At different time points after release into S phase, change culture medium to RPE1 medium containing 100 μM EU. 4. After 1 or 2 hr of labeling nascent transcripts with EU, discard EU medium and wash cells once with PBS, then collect cells in 1.2 ml TRIzol using cell lifter. EU labeling has been reported to occur in as little as 10 min in vivo (Jao and Salic, 2008).
5. Flash-freeze samples in liquid nitrogen and store them at −80°C.
Whole cell total RNA extraction 6. Add 0.5 ml chloroform to samples collected in 1.25 ml TRIzol. 7. Mix well and incubate 10 min at room temperature. 8. Centrifuge samples 15 min at 12,000 × g, 4°C. 9. Remove aqueous phase (900 μl) and place in a new tube. 10. Add isopropanol (900 μl; equal volume relative to aqueous phase) to aqueous phase from step 9 and incubate on ice 5 min. 11. Centrifuge samples 15 min at 12,000 × g, 4°C to precipitate total RNA. 12. Wash RNA pellet with 80% ethanol. 13. Centrifuge 5 min at 12,000 × g, 4°C. Isolation of Nascent Transcripts
14. Air dry pellet 5 min.
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15. Resuspend dried pellet in 44 μl nuclease-free water.
DNase treatment 16. Add 5 μl 10× Baseline-ZERO reaction buffer to the samples from step 15 and vortex. 17. Add 1 μl Baseline-ZERO DNase and mix gently by pipetting; do not vortex. 18. Incubate at 37°C 30 min. 19. Purify reaction with RNA Clean & Concentrator kit, according to manufacturer’s protocol. 20. Elute sample in 25 μl nuclease-free water.
Biotinylation of EU-labeled RNA with click chemistry 21. Prepare fresh 250 mM sodium L-ascorbate. 22. Add the following sequentially to the RNA sample (1 to 5 μg) from step 20 using a pipet. The reaction can be scaled up; the example reaction below has a final reaction volume of 60 μl.
6 μl 1 M Tris·HCl, pH 7.5 (final concentration: 100 mM Tris·HCl, pH 7.5) 6 μl acetonitrile (final concentration: 10% acetonitrile) 3.6 μl 250 mM sodium L-ascorbate (final concentration: 15 mM sodium L-ascorbate) 0.6 μl 50 mM biotin-dPEG 7-azide (final concentration: 0.5 mM Biotin-dPEG 7-azide) 6 μl 1 M PMDTA (final concentration: 100 mM PMDTA) 0.6 μl 100 mM CuSO4 (final concentration: 1 mM CuSO4 ) Just before addition of CuSO4 -ligand complex, it is beneficial to degas the reaction with argon or nitrogen. There are several different ligand alternatives to PMDTA, such as tris(benzyltriazolylmethyl)amine (TBTA) and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA). We chose PMDTA because it is commercially available in high purity. Importantly, it is water soluble. Unlike the most commonly used ligand, TBTA, PMDTA does not precipitate in an aqueous environment. Having an efficient ligand that remains in solution is crucial because otherwise RNA becomes exposed to copper oxidation.
23. Mix well and incubate in the dark 30 min at 45°C in a thermomixer (750 rpm). 24. Clean up the reaction with ethanol precipitation, bring reaction volume to 300 μl with water; add 9 μl glycogen (5 mg/ml), 30 μl 3 M sodium acetate and 900 μl 100% ethanol. Incubate samples at −80°C for 2 hr. 25. Centrifuge 30 min at 16,000 × g, 4°C to precipitate RNA. 26. Wash pellet once with 80% ethanol. 27. Centrifuge 10 min at 16,000 × g, 4°C. 28. Air dry pellet and resuspend in 50 μl TNE0.2. Check yield with NanoDrop. Biotinylated RNA samples can be stored at −80°C up to a week until used in the streptavidin pull-down step. Long-term storage is not recommended. Preparation and Analysis of RNA
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Block streptavidin beads 29. One day before the streptavidin isolation step, block 50 μl Dynabeads MyOne Streptavidin C1 beads per 1 to 2.5 μg click-modified RNA sample (from step 28). 30. Wash beads once with TNE2.0 5 min at room temperature. Place tubes in magnetic stand 2 min at room temperature and remove wash buffer. 31. Wash beads once with 500 μl wash buffer 65 in a thermomixer at 65°C while mixing vigorously for 10 min. Place tubes in magnetic stand 2 min at room temperature and remove wash buffer with a pipet. 32. Wash beads twice with 500 μl TNE0.2 buffer at room temperature. Place tubes on magnetic stand 2 min at room temperature and remove wash buffer with a pipet. 33. Resuspend beads in 1× TNE bead-blocking buffer and block beads 24 hr at 4°C while rotating at high setting.
Isolate nascent transcripts with streptavidin beads 34. Denature biotinylated RNA at 65°C, 5 min, then quickly chill on ice. 35. Add 1 μl RNasin Plus RNase inhibitor and remove 0.5 μl to store as input. Store input at −80°C until all samples are ready for first strand synthesis step. 36. Resuspend blocked beads in 250 μl TNE0.2. 37. Add 50.5 μl denatured click-modified RNA to the beads. 38. Incubate blocked beads and biotin click-modified samples in a thermomixer at 18°C while mixing vigorously (1100 rpm) in the dark for 20 min. 39. Wash beads once with 700 μl TNE2.0 at room temperature. 40. Wash beads once with 500 μl wash buffer 65 while mixing vigorously (1100 rpm) in a thermomixer at 65°C for 10 min; immediately apply to magnetic stand. 41. Wash beads twice with 500 μl TNE0.2 at room temperature. 42. Resuspend beads in 10 μl nuclease-free water.
Detection of gene-specific nascent RNA enrichment 43. To synthesize cDNA on beads with SuperScript VILO cDNA synthesis kit, add the following to a PCR tube: 4 μl 5× Vilo Reaction mix 2 μl 10× SuperScript Enzyme mix 12 μl beads in water from step 42 Make up reaction volume to 20 μl with RNAse-free water. Mix gently by flicking. 44. Incubate 10 min at 25°C, then 60 min at 42°C, and then 5 min at 85°C in a thermocycler.
Purify reaction products 45. Add 6.5 μl 0.5 M EDTA and 6.5 μl 1 N NaOH; mix well and incubate at 65°C for 15 min. Then add 17 μl 1 M HEPES, pH 7.5. Samples can be snap-frozen after this step. Isolation of Nascent Transcripts
46. Separate beads using a magnetic stand and transfer samples into YM-10 Amicon columns. Add 500 μl nuclease-free water and centrifuge at 14,000 × g for 10 min at room temperature.
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47. Add another 500 μl nuclease-free water to the columns and centrifuge at 14,000 × g for 12 min. 48. Place columns in a clean tube, invert, and spin at 10,000 × g for 1 min to collect samples. The collected sample volume should be 30 μl.
49. Use isolated cDNA from step 48 to detect gene of interest with PCR/qPCR.
Strand-specific detection of nascent RNA enrichment genome wide Steps 50 to 56 provide a genome-wide alternative to the gene-type-specific method for detection of nascent transcripts. 50. Remove ribosomal RNA (rRNA) with Ribo-Zero Gold Magnetic kit following the kit manufacturer’s instructions. rRNA removal step must be done before you perform the click reaction!
51. Clean up and concentrate rRNA-depleted samples with RNA Clean & Concentrator kit, according to the manufacturer’s protocol. Repeat steps 21 through 42. RNA fragmentation: mix 5 μl First Strand buffer with 10 μl rRNA-depleted nascent RNA, enriched through biotin-streptavidin interaction (from step 42). Incubate at 94°C for 5 min in a thermocycler, then quickly chill samples on ice. 52. For first strand synthesis, add the following to the fragmented RNA: 1.5 μl 100 mM DTT (SuperScript III, Life Technologies) 1.25 μl Random Primers (Life Technologies, cat. no. 48190011) Incubate at 65°C for 3 min, chill on ice, then add the following: 1.5 μl 10 mM dNTPs 0.75 μl RNasin 1.5 μl 100 mM DTT 8 μl nuclease-free water 1.5 μl SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080-044) Incubate at 25°C for 10 min, 50°C for 1 hr, 70°C 15 min, 4°C forever in a thermocycler. 53. Purify reaction products using Amicon YM10 columns as described in steps 46 to 48; eluate should be 50 μl. 54. For second strand synthesis, add the following to the samples from previous step: 2 μl 5× First Strand buffer 0.5 μl 100 mM MgCl2 1 μl 100 mM DTT 15 μl 5× Second Strand buffer (Life Technologies, cat. no. 10812-014) 2 μl 10 mM dNTP mix (use dUTP instead of dTTP for strand-specific sequencing) 0.5 μl E. coli DNA ligase (New England BioLabs, cat. no. M205S) 2 μl E. coli DNA polymerase (New England BioLabs, cat. no. M209L) 0.5 μl E. coli RNase H (New England BioLabs, cat. no. M0297S) Incubate at 16°C for 2.5 hr.
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55. Purify reaction products using 1:1.8 reaction volume/Ampure XP beads; add 135 μl Ampure XP beads and mix by pipetting up and down. Place tubes in magnetic stand for 10 min to capture beads. Wash beads with freshly prepared 80% ethanol without disturbing the bead pellet. Do not remove tubes from magnetic stand.
Wait 2 min and aspirate ethanol. Repeat the wash step. Air dry pellet for 5 to 6 min. It is important not to over dry beads as it lowers elution efficiency.
Resuspend beads in 36 μl nuclease-free water, pipet up and down, and incubate at room temperature for 5 min. Place tubes in magnetic stand for 5 min to capture beads. Transfer eluted DNA to a new tube. 56. Continue with sequencing library construction as described earlier (Bowman et al., 2013; UNIT 4.21, Podnar et al., 2014). 57. UDG treatment for strand specificity: add 2 μl UDG to 30 μl eluted DNA and incubate at 37°C for 30 min. This step should be done just before sequencing library amplification PCR, right after the adapter ligation step. SUPPORT PROTOCOL
ASSESSING CLICK EFFICIENCY WITH RNA DOT-BLOT RNA dot/slot blotting is a simple technique that allows hybridization and analysis of unfractionated RNA. It is similar to DNA dot blotting except for the denaturation step of samples prior to their immobilization on the membrane. The protocol described here is modified from Dolken et al., 2008 (also see UNIT 4.9; Brown et al., 2004).
Additional Materials (also see Basic Protocol) Formamide (Sigma-Aldrich, cat. no. F9037) 20× SSC (Ambion, cat. no. AM9763) Pierce High Sensitivity Streptavidin-HRP (Life Technologies, cat. no. 21134) Nucleic Acid Detection blocking buffer (Thermo Scientific, cat. no. 89880 A) Binding solution (see recipe) Streptavidin-HRP wash buffer (see recipe) Zeta-Probe membrane (Bio-Rad, cat. no. 162-0153) Diethyl pyrocarbonate (DEPC)-treated water (see recipe) Tris-buffered saline and Tween 20 (TBST) Nuclease-free BSA (Sigma-Aldrich, cat. no. B2518) Whatman 3 MM paper Bio-Dot apparatus (Bio-Rad) UV crosslinker (UV Stratalinker 2400) Prepare membrane and assemble manifold for transfer 1. Clean dot-blot manifold with RNaseZap solution and rinse well with DEPC-treated water. 2. Cut membrane to the same size as the manifold and pre-wet membrane in a clean glass tray with 10× SSC for 5 to 10 min. Handle membrane only with RNaseZap-cleaned, blunt-ended forceps. Avoid touching membrane even with gloves on. Isolation of Nascent Transcripts
3. Assemble the membrane and the manifold according to the manufacturer’s instructions. Mark membrane for orientation.
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4. Fill each well with 500 μl nuclease-free water. Check wells for even suction and make sure there are no air bubbles in the wells or air leaks in the manifold assembly. Be careful with the amount of vacuum applied because too much suction can damage the membrane. Use manifold suction setting 3.
Denature RNA samples and immobilize them on the membrane 5. Denature biotinylated RNA at 65°C for 10 min, then chill sample on ice. 6. Add 20 μl 10× binding solution to the denatured and cooled samples. Bring volume up to 200 μl with nuclease-free water. Mix well and transfer mixture to the wells. Fill empty wells with same amount of 2× SSC, and then apply gentle suction. 7. Wash wells with 500 μl 1× binding solution buffer and apply suction. 8. Disassemble manifold; air dry the membrane on clean Whatman 3 MM paper. 9. UV crosslink membrane in UV Stratalinker 2400 using the auto-crosslink setting.
Detect streptavidin-HRP 10. Block membrane with 12 ml Nucleic Acid Detection blocking buffer for 30 min at room temperature on an orbital shaker. 11. Dilute Pierce High-Sensitivity Streptavidin-HRP 1:100 with 12 ml 5% nuclease-free BSA in TBST and incubate 15 min at room temperature on an orbital shaker. 12. Wash membrane once with 1:10 dilution of Nucleic Acid Detection blocking buffer/water 5 min at room temperature. 13. Wash twice with streptavidin-HRP wash buffer 10 min at room temperature. 14. Wash once with TBST 5 min at room temperature. 15. Detect biotin-streptavidin-HRP complex with enhanced chemiluminescence.
REAGENTS AND SOLUTIONS Use nuclease-free water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2.
Binding solution, 10× 100 mM NaOH 10 mM EDTA Store solution up to one year at −20°C Diethyl pyrocarbonate (DEPC)-treated water Add 0.1% diethyl pyrocarbonate to water and mix well. Incubate at 37°C for 2 hr. Autoclave solution to destroy diethyl pyrocarbonate as it may modify RNA if present in the solution. Store at room temperature indefinitely. Mimosine stock solution Dissolve 79 mg mimosine in 6 ml 0.1 N sodium hydroxide (cell culture tested). After mimosine has completely dissolved, add 4 ml DMEM/F-12 medium. Filter and sterilize solution. Aliquot and store at −80°C indefinitely RPE1 medium 500 ml Advanced DMEM/F-12 (Life Technologies, cat. no. 12634-010) 50 ml FBS continued
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5 ml Glutamax (Life Technologies, cat. no. 35050-061) 5 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 17.3 ml 7.5% sodium bicarbonate (Sigma-Aldrich, cat. no. S5761) Mix well, filter, and sterilize Store solution at 4°C for up to 3 months Streptavidin-HRP wash buffer 10× stock solution contains: 100 mM Tris·Cl, pH 7.6 (APPENDIX 2) 100 mM NaCl 10 mM EDTA Add 0.05% Tween 20 fresh to 1× solution each time Store 10× stock solution at room temperature up to 6 months TNE bead-blocking buffer 10× stock solution contains: 100 mM Tris·Cl, pH 7.5 (APPENDIX 2) 10 mM EDTA, pH 8.0 2 M NaCl 20 mg/ml nuclease-free BSA 10 μg/ml poly(deoxyinosinic-deoxycytidylic) acid, sodium salt (polydI-dC) Aliquot and store up to a year at −20°C TNE0.2 10 mM Tris·Cl, pH 7.4 (APPENDIX 2); 1 mM EDTA, pH 8.0; and 200 mM NaCl in nuclease-free water TNE2.0 10 mM Tris·Cl, pH 7.4 (APPENDIX 2); 1 mM EDTA, pH 8.0; and 2 M NaCl in nuclease-free water Store up to a year at room temperature Wash buffer 65 100 mM Tris·Cl, pH 7.4 (APPENDIX 2) 10 mM EDTA 1 M NaCl 0.1% Tween 20 Mix first three items. Store buffer up to a year at room temperature. Add 0.1% Tween 20 fresh each time just before use. COMMENTARY Background Information
Isolation of Nascent Transcripts
Copper-catalyzed azide-alkyne cycloaddition (CuAAC), also known as a “click” reaction (Kolb et al., 2001; Rostovtsev et al., 2002) enables efficient conjugation of azidecontaining molecules to ethynyl-labeled nucleic acids both in vitro (Paredes and Das, 2010) and in vivo (Salic and Mitchison, 2008). These conjugated molecules can then be selectively enriched via affinity purification through biotin-streptavidin interaction. In vivo incorporation of the EU into the nascent transcripts and their microscopic detection via coppercatalyzed cycloaddition (click reaction) of flu-
orescent azides have been described (Jao and Salic, 2008). Standard gene expression analyses on total cellular RNA are only capable of profiling steady-state levels of RNA at a given time, but lack the ability to measure true transcriptional response. In addition, the results are obscured by different RNA decay rates, and are especially biased against transcripts with long half-lives (Friedel and D¨olken, 2009). To circumvent some of these limitations, earlier RNA-decay studies used drugs to inhibit transcription (Bernstein et al., 2002; Raghavan et al., 2002) and utilized nuclear run-on
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assays to detect nascent transcripts in a genespecific manner (Hirayoshi and Lis, 1999) or a genome-wide manner with radiolabeled nucleotides (Garcıa-Martınez et al., 2004) or the nucleotide analog 5-bromouridine 5 triphosphate (BrUTP; Core et al., 2008). These approaches are both technically demanding and, more importantly, experimentally biased as they are cell invasive. In vivo labeling with 5 -bromouridine (BrU) and isolation of nascent transcripts with anti-BrU antibody has also been tested (Ohtsu et al., 2008). While this approach is technically less demanding compared to nuclear run-on-based assays, it is disadvantageous due to BrU toxicity and the relative inefficiency of capturing RNAs with low uridine content. Thiol-containing nucleosides are also known to incorporate into nascent transcripts. While some versions of thiol-modified nucleoside analogs show toxicity, 4-thiouridine (4sU) has been shown to be devoid of cytotoxic effects (Dolken et al., 2008). It was used to profile genome-wide mRNA synthesis and decay rates in yeast (Miller et al., 2011) and mammals (Rabani et al., 2011). Hence, in many aspects, metabolic labeling of nascent RNA with 4sU and EU is similar except for the biotinylation step (Zeiner et al., 2008). One advantage of biotinylation with click chemistry is that it requires much less material in terms of both RNA and biotin. Also, it is a much more rapid reaction than chemical biotinylation of 4sU (30 min versus 3 hr). In addition, unlike 4sU, EU-click chemistry has the potential to be used in whole cells to isolate nascent RNA-associated factors.
Critical Parameters RNA quality High quality RNA is essential for the success of each and every step of this protocol. In particular, RNA integrity should be checked after the copper-catalyzed azide-alkyne cycloaddition step. A good check is to run two reactions for each sample where one reaction is run without copper as a control, to check ligand efficiency in stabilizing copper and to assess copper-related degradation. Pre-mixing ligand (PMDTA or THPTA) and copper (CuSO4 ), then adding the combination to the reaction is beneficial to further avoid copper-catalyzed oxidation of RNA. Another way of reducing the risk of coppermediated RNA oxidation is to degas the reaction by bubbling argon or nitrogen gas.
Click reaction efficiency The basic CuAAC reaction requires copper ions in the +1 oxidation state. Instead of using Cu(I) directly, Cu(II) salts reduced to Cu(I) by ascorbic acid during the reaction are preferred to avoid undesired by-products (Rostovtsev et al., 2002). Sodium ascorbate not only allows a safer reactive copper (i.e., allows a more controlled reaction environment with less oxidative stress) but also extends the half-life of the reactive Cu(I) species in the reaction. It is crucial to maintain high Cu(I) levels at all times during the click reaction for high yield. Using at least a five-fold excess of ascorbate over Cu(II) salt is important for reaction efficiency. Also, sodium ascorbate needs to be prepared fresh. Using a co-solvent such as acetonitrile is important for both copper coordination and RNA integrity. Including a ligand such as PMDTA in the reaction is particularly important as it significantly accelerate the reaction. Another factor that contributes to the reaction rate is the reaction temperature as the click reaction occurs more efficiently at higher temperatures (Meldal and Tornøe, 2008). Control samples Including reactions lacking copper or without EU labeling act as good controls, to allow control of RNA integrity and also to assess pull-down specificity. Next-generation RNA sequencing If nascent RNA will be analyzed with highthroughput sequencing then samples should be depleted of rRNA before biotinylation with the click reaction because rRNA-depletion kits utilize streptavidin-coated beads.
Troubleshooting The RNA yield with ethanol precipitation after the click reaction (Basic Protocol, step 28) gives the first indication of RNA integrity. Unless there is RNA degradation there should not be any difference in the amount of starting material and precipitated sample. More reliable assessment of RNA integrity is provided by comparing the click reaction sample and its control reaction sample (which does not contain CuSO4 ) with RT-PCR for a known expressed target. For example, less PCR product from the click reaction than from the (control) reaction in the absence of copper indicates a failure in copper stabilization and/or an oxidized reaction environment. Use of freshly
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prepared PMDTA and fresh sodium ascorbate is recommended. Also, increasing the time argon is bubbled through the reaction to degas the mixture can be beneficial. After the streptavidin pull-down step, control samples (i.e, samples from reactions run in the absence of CuSO4 or EU) should not produce any detectable PCR product. Any PCR products from those samples suggest a nonspecific pull down (false positive). The bead-blocking step is crucial to avoid nonspecific pull down. The bead-blocking step and/or 65°C wash step can be extended to avoid this result.
Anticipated Results This protocol allows isolation of nascent transcripts using the desired conditions depending on the experimental design. RT-qPCR after nascent RNA pull down can be used to examine whether a gene of interest is newly synthesized. Alternatively, nextgeneration sequencing after nascent transcript isolation will provide a genome-wide map of newly synthesized RNAs. When labeling cellcycle-specific nascent transcripts, replicationdependent canonical histones serve as a useful positive control for S phase and can be used as a negative control for M phase. Another control is the Xist transcript as it is maintained at the same levels throughout the cell cycle.
Time Considerations Cell cycle synchronization, EU labeling and sample collection can be completed in 2 days. Total RNA isolation from those samples and DNase treatment takes about 2 hr. If pulled down RNA will be analyzed with highthroughput sequencing then samples should be depleted of rRNA. This additional step takes about an hour. Biotinylation with the click reaction, followed by sample precipitation and streptavidin pull down of nascent transcripts, requires 4 to 5 hr. The cDNA synthesis step should be performed immediately once nascent RNAs are pulled down, which takes about 2 hr, then the RNA of interest can be searched in the nascent RNA pull-down material using a regular PCR reaction in about 1 hr.
Acknowledgements Isolation of Nascent Transcripts
Ozlem Yildirim is an HHMI Fellow of The Damon Runyon Cancer Research Foundation (DRG-2156-13). The author would like to acknowledge Kingston Lab members for stimulating discussions.
Literature Cited Bernstein, J.A., Khodursky, A.B., Lin, P.-H., LinChao, S., and Cohen, S.N. 2002. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc. Natl. Acad. Sci. U.S.A. 99:9697-9702. Bowman, S.K., Simon, M.D., Deaton, A.M., Tolstorukov, M., Borowsky, M.L., and Kingston, R.E. 2013. Multiplexed Illumina sequencing libraries from picogram quantities of DNA. BMC Genomics 14:466. Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by Northern and slot blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1-4.9.19. Core, L.J., Waterfall, J.J., and Lis, J.T. 2008. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322:1845-1848. Curanovic, D., Cohen, M., Singh, I., Slagle, C.E., Leslie, C.S., and Jaffrey, S.R. 2013. Global profiling of stimulus-induced polyadenylation in cells using a poly(a) trap. Nat. Chem. Biol. 9:671-673. Dolken, L., Ruzsics, Z., Radle, B., Friedel, C.C., Zimmer, R., Mages, J., Hoffmann, R., Dickinson, P., Forster, T., Ghazal, P., and Koszinowski, U.H. 2008. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14:1959-1972. Friedel, C.C. and D¨olken, L. 2009. Metabolic tagging and purification of nascent RNA: Implications for transcriptomics. Mol. Biosyst. 5:12711278. Garcıa-Martınez, J., Aranda, A., and P´erez-Ortın, J.E. 2004. Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms. Mol. Cell 15:303313. Hirayoshi, K. and Lis, J.T. 1999. Nuclear run-on assays: Assessing transcription by measuring density of engaged RNA polymerases. Methods Enzymol. 304:351-362. Jao, C.Y. and Salic, A. 2008. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. U.S.A. 105:15779-15784. Kolb, H.C., Finn, M.G., and Sharpless, K.B. 2001. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40:2004-2021. Meldal, M. and Tornøe, C.W. 2008. Cucatalyzed azide-alkyne cycloaddition. Chem. Rev. 108:2952-3015. Miller, C., Schwalb, B.O.R., Maier, K., Schulz, D., D¨umcke, S., Zacher, B., Mayer, A., Sydow, J., Marcinowski, L., Martin, D.E., Tresch, A., and Cramer, P. 2011. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Mol. Sys. Biol. 7:458. Ohtsu, M., Kawate, M., Fukuoka, M., Gunji, W., Hanaoka, F., Utsugi, T., Onoda, F., and Murakami, Y. 2008. Novel DNA microarray
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system for analysis of nascent mRNAs. DNA Res. 15:241-251. Paredes, E. and Das, S.R. 2010. Click chemistry for rapid labeling and ligation of RNA. ChemBioChem 12:125-131. Podnar, J., Deiderick, H., Huerta, G., and HunickeSmith, S. 2014. Next-generation sequencing RNA-Seq library construction. Curr. Protoc. Mol. Biol. 106:4.21.1-4.21.19. Rabani, M., Levin, J.Z., Fan, L., Adiconis, X., Raychowdhury, R., Garber, M., Gnirke, A., Nusbaum, C., Hacohen, N., Friedman, N., Amit, I., and Regev, A. 2011. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat. Biotechnol. 29:436-442. Raghavan, A., Ogilvie, R.L., Reilly, C., Abelson, M.L., Raghavan, S., Vasdewani, J., Krathwohl,
M., and Bohjanen, P.R. 2002. Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res. 30:5529-5538. Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:25962599. Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415-2420. Zeiner, G.M., Cleary, M.D., Fouts, A.E., Meiring, C.D., Mocarski, E.S., and Boothroyd, J.C. 2008. RNA analysis by biosynthetic tagging using 4thiouracil and uracil phosphoribosyltransferase. Methods Mol. Biol. 419:135-146.
Preparation and Analysis of RNA
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UNIT 4.24
Ozlem Yildirim1,2 1
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 2 Department of Genetics, Harvard Medical School, Boston, Massachusetts
Steady-state levels of cellular RNA are determined by both transcriptional rate and RNA half-life. Commonly used methods for transcriptional analysis are only capable of profiling total RNA and do not distinguish changes in synthesis and decay rates. Hence, a better understanding of the temporal dynamics of cellular response for a given condition at the transcriptional level requires techniques for the analysis of nascent transcripts. Here we describe a protocol that allows isolation of nascent transcripts with a copper-catalyzed azide-alkyne C 2015 by cycloaddition (CuAAC) also known as a click chemistry reaction. John Wiley & Sons, Inc. Keywords: nascent RNA r metabolic labeling r click chemistry
How to cite this article: Yildirim, O. 2015. Isolation of nascent transcripts with click chemistry. Curr. Protoc. Mol. Biol. 111:4.24.1-4.24.13. doi: 10.1002/0471142727.mb0424s111
INTRODUCTION To better understand the cellular response to a stimulus or a condition it is important to determine the acute changes in gene expression programs upon that stimulus. This requires a method that can discriminate the nascent transcripts from total cellular RNA, as differences in transcript half-lives of different genes greatly obscure the true transcriptional response. In addition to its use for studying temporal transcriptional activity, such a protocol can be used for analysis of RNA decay rates. Recently, different approaches, which mainly utilize the metabolic tagging of nascent RNAs with nucleoside analogs, have emerged (Core et al., 2008; Dolken et al., 2008; Rabani et al., 2011; Curanovic et al., 2013). Similarly, the protocol in this unit utilizes metabolic incorporation of nucleoside analog 5-ethynyl uridine (EU) into newly synthesized RNAs. It has been shown in vivo that EU incorporation is specific to RNA molecules and it does not nonspecifically label cellular DNA (Jao and Salic, 2008). Then azide-containing biotin molecules are covalently attached to the alkyne-modified uridine-containing transcripts with the assistance of copper catalysis in vitro (Paredes and Das, 2010). Biotinylated EU residues in the nascent transcripts allow the efficient isolation with streptavidin-coated magnetic beads. The Basic Protocol describes labeling and isolation of newly synthesized RNAs from synchronized cells. These RNAs can be sequenced to determine the spectrum of RNAs that are synthesized. It is frequently useful to determine the efficiency of incorporation of the EU label prior to performing further analysis of the sample. The Support Protocol describes how to accomplish this procedure.
Preparation and Analysis of RNA Current Protocols in Molecular Biology 4.24.1-4.24.13, July 2015 Published online July 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142727.mb0424s111 C 2015 John Wiley & Sons, Inc. Copyright
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ISOLATION OF CELL-CYCLE-SPECIFIC NASCENT TRANSCRIPTS WITH CLICK CHEMISTRY This protocol allows isolation of newly synthesized RNAs at any stage of the cell cycle. First, cells are synchronized at the G1/S border. After release into the S phase, the cell culture medium is changed to EU-containing medium for 1 or 2 hr to label nascent RNA. Labeled RNA is then isolated by biotin-streptavidin interaction (Fig. 4.24.1).
Materials
Isolation of Nascent Transcripts
Cultured cells (e.g., hTERT immortalized RPE1 cells; 2 × 106 to 5 × 106 cells for each experiment) RPE1 medium (see recipe) 5-Ethynyl uridine (Berry & Associates, cat. no. PY7563) TRIzol reagent (Life Technologies, cat. no. 15596-026) Nuclease-free water (Ambion, cat. no. AM9932) Chloroform (Sigma-Aldrich, cat. no. C2432) 100% and 80% ethanol Isopropanol (Sigma-Aldrich, cat. no. I9516) 10× Baseline-ZERO reaction buffer Baseline-ZERO DNase (Epicentre, cat. no. DB0715K) RNA Clean & Concentrator kit (Zymo Research, cat. no. R1015) Acetonitrile (Sigma-Aldrich, cat. no. 34998) 1 M Tris·HCl, pH 7.5 Sodium-L-ascorbate (Sigma-Aldrich) Copper sulfate (CuSO4 ; Sigma-Aldrich, cat. no. 451657) Biotin-dPEG 7-azide (Quanta BioDesign, cat. no. 10825) N,N,N ,N ,N -pentamethyldiethylenetriamine (PMDTA; Sigma-Aldrich, cat. no. 369497) L-Mimosine (Sigma-Aldrich, cat. no. M0253) Glycogen (Ambion, cat. no. AM9510) Dynabeads MyOne Streptavidin C1 magnetic beads (Life Technologies, cat. no. 65001) 3 M sodium acetate, pH 5.5 (Ambion, cat. no. AM9740) RNasin Plus Rnase inhibitor (Promega) Ribo-Zero Gold Magnetic kit (Epicentre, cat. no. MRZG12324) SuperScript VILO cDNA synthesis kit (Life Technologies, cat. no. 11754-050) RNAse-free water 0.5 M EDTA 1 N NaOH 1 M HEPES 100 mM DTT (Life Technologies, cat. no. 18080-044) SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080-044) Random primers (Life Technologies, cat. no. 48190011) 10 mM dNTPs First Strand buffer (Life Technologies, cat. no. 18080-044) 5× Second Strand buffer (Life Technologies, cat. no. 10812-014) E. coli DNA ligase (New England BioLabs, cat. no. M0205S) E. coli DNA polymerase (New England BioLabs, cat. no. M0209L) E. coli RNase H (New England BioLabs, cat. no. M0297S) Uracil DNA glycosylase (UDG; New England BioLabs, cat. no. M0280S) TNE bead-blocking buffer (see recipe) TNE2.0 (see recipe) TNE0.2 (see recipe) Wash buffer 65 (see recipe)
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Figure 4.24.1 (A) Schematic of experimental design of the in vivo assay. (B) Nascent transcripts are labeled with EU at different time points into S phase. RNA is isolated and conjugated to UV-cleavable biotin-azide via a click reaction. As expected, S phase-specific histone H2B was enriched in EU-labeled samples but not in unlabeled samples. (C) Xist RNA which is known to exist throughout the cell cycle is enriched in EU-labeled samples but not in unlabeled samples. Preparation and Analysis of RNA
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RNaseZap (Ambion, cat. no. AM9780) Amicon YM-10 Ultra-0.5 Centrifugal Filter Unit (EMD Millipore, cat. no. UFC5010BK) Ampure XP beads (Beckman Coulter Genomics, cat. no. A63881) Dyna Mag magnetic stand (Life Technologies, cat. no. 12321D) Cell lifter (Corning, cat. no. 3008) Low-binding, nuclease-free 1.7-ml tubes (Eppendorf, cat. no. 022431021) Thermomixer (Eppendorf, cat. no. 022670107) Rotator (Thomas Scientific, cat. no. 1217H25) Thermocycler NOTE: All solutions must be prepared with Rnase-free water.
Synchronization and metabolic labeling of cultured cells with EU 1. Grow hTERT immortalized RPE1 cells to 75% to 80% confluence. Trypsinize and plate 3 × 106 cells per 15-cm dish in RPE1 medium with a final concentration of 450 μM mimosine to arrest cells at G1/S border. This protocol can work with other cell types once the cell type-specific synchronization conditions are determined. We chose hTERT-RPE1 cells for two main reasons: first, they are diploid immortalized cells; second, they can be very efficiently synchronized at the G1/S border of the cell cycle.
2. After 21 hr of mimosine synchronization, wash cells twice with 15 ml PBS and release them into S phase in regular culture medium. For hTERT-RPE1 cells it takes approximately 3 hr for 20% of the population to enter S phase. We recommend testing different timing and conditions followed by fluorescenceactivated cell sorting (FACS) to determine the best conditions for desired synchronization.
3. At different time points after release into S phase, change culture medium to RPE1 medium containing 100 μM EU. 4. After 1 or 2 hr of labeling nascent transcripts with EU, discard EU medium and wash cells once with PBS, then collect cells in 1.2 ml TRIzol using cell lifter. EU labeling has been reported to occur in as little as 10 min in vivo (Jao and Salic, 2008).
5. Flash-freeze samples in liquid nitrogen and store them at −80°C.
Whole cell total RNA extraction 6. Add 0.5 ml chloroform to samples collected in 1.25 ml TRIzol. 7. Mix well and incubate 10 min at room temperature. 8. Centrifuge samples 15 min at 12,000 × g, 4°C. 9. Remove aqueous phase (900 μl) and place in a new tube. 10. Add isopropanol (900 μl; equal volume relative to aqueous phase) to aqueous phase from step 9 and incubate on ice 5 min. 11. Centrifuge samples 15 min at 12,000 × g, 4°C to precipitate total RNA. 12. Wash RNA pellet with 80% ethanol. 13. Centrifuge 5 min at 12,000 × g, 4°C. Isolation of Nascent Transcripts
14. Air dry pellet 5 min.
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15. Resuspend dried pellet in 44 μl nuclease-free water.
DNase treatment 16. Add 5 μl 10× Baseline-ZERO reaction buffer to the samples from step 15 and vortex. 17. Add 1 μl Baseline-ZERO DNase and mix gently by pipetting; do not vortex. 18. Incubate at 37°C 30 min. 19. Purify reaction with RNA Clean & Concentrator kit, according to manufacturer’s protocol. 20. Elute sample in 25 μl nuclease-free water.
Biotinylation of EU-labeled RNA with click chemistry 21. Prepare fresh 250 mM sodium L-ascorbate. 22. Add the following sequentially to the RNA sample (1 to 5 μg) from step 20 using a pipet. The reaction can be scaled up; the example reaction below has a final reaction volume of 60 μl.
6 μl 1 M Tris·HCl, pH 7.5 (final concentration: 100 mM Tris·HCl, pH 7.5) 6 μl acetonitrile (final concentration: 10% acetonitrile) 3.6 μl 250 mM sodium L-ascorbate (final concentration: 15 mM sodium L-ascorbate) 0.6 μl 50 mM biotin-dPEG 7-azide (final concentration: 0.5 mM Biotin-dPEG 7-azide) 6 μl 1 M PMDTA (final concentration: 100 mM PMDTA) 0.6 μl 100 mM CuSO4 (final concentration: 1 mM CuSO4 ) Just before addition of CuSO4 -ligand complex, it is beneficial to degas the reaction with argon or nitrogen. There are several different ligand alternatives to PMDTA, such as tris(benzyltriazolylmethyl)amine (TBTA) and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA). We chose PMDTA because it is commercially available in high purity. Importantly, it is water soluble. Unlike the most commonly used ligand, TBTA, PMDTA does not precipitate in an aqueous environment. Having an efficient ligand that remains in solution is crucial because otherwise RNA becomes exposed to copper oxidation.
23. Mix well and incubate in the dark 30 min at 45°C in a thermomixer (750 rpm). 24. Clean up the reaction with ethanol precipitation, bring reaction volume to 300 μl with water; add 9 μl glycogen (5 mg/ml), 30 μl 3 M sodium acetate and 900 μl 100% ethanol. Incubate samples at −80°C for 2 hr. 25. Centrifuge 30 min at 16,000 × g, 4°C to precipitate RNA. 26. Wash pellet once with 80% ethanol. 27. Centrifuge 10 min at 16,000 × g, 4°C. 28. Air dry pellet and resuspend in 50 μl TNE0.2. Check yield with NanoDrop. Biotinylated RNA samples can be stored at −80°C up to a week until used in the streptavidin pull-down step. Long-term storage is not recommended. Preparation and Analysis of RNA
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Block streptavidin beads 29. One day before the streptavidin isolation step, block 50 μl Dynabeads MyOne Streptavidin C1 beads per 1 to 2.5 μg click-modified RNA sample (from step 28). 30. Wash beads once with TNE2.0 5 min at room temperature. Place tubes in magnetic stand 2 min at room temperature and remove wash buffer. 31. Wash beads once with 500 μl wash buffer 65 in a thermomixer at 65°C while mixing vigorously for 10 min. Place tubes in magnetic stand 2 min at room temperature and remove wash buffer with a pipet. 32. Wash beads twice with 500 μl TNE0.2 buffer at room temperature. Place tubes on magnetic stand 2 min at room temperature and remove wash buffer with a pipet. 33. Resuspend beads in 1× TNE bead-blocking buffer and block beads 24 hr at 4°C while rotating at high setting.
Isolate nascent transcripts with streptavidin beads 34. Denature biotinylated RNA at 65°C, 5 min, then quickly chill on ice. 35. Add 1 μl RNasin Plus RNase inhibitor and remove 0.5 μl to store as input. Store input at −80°C until all samples are ready for first strand synthesis step. 36. Resuspend blocked beads in 250 μl TNE0.2. 37. Add 50.5 μl denatured click-modified RNA to the beads. 38. Incubate blocked beads and biotin click-modified samples in a thermomixer at 18°C while mixing vigorously (1100 rpm) in the dark for 20 min. 39. Wash beads once with 700 μl TNE2.0 at room temperature. 40. Wash beads once with 500 μl wash buffer 65 while mixing vigorously (1100 rpm) in a thermomixer at 65°C for 10 min; immediately apply to magnetic stand. 41. Wash beads twice with 500 μl TNE0.2 at room temperature. 42. Resuspend beads in 10 μl nuclease-free water.
Detection of gene-specific nascent RNA enrichment 43. To synthesize cDNA on beads with SuperScript VILO cDNA synthesis kit, add the following to a PCR tube: 4 μl 5× Vilo Reaction mix 2 μl 10× SuperScript Enzyme mix 12 μl beads in water from step 42 Make up reaction volume to 20 μl with RNAse-free water. Mix gently by flicking. 44. Incubate 10 min at 25°C, then 60 min at 42°C, and then 5 min at 85°C in a thermocycler.
Purify reaction products 45. Add 6.5 μl 0.5 M EDTA and 6.5 μl 1 N NaOH; mix well and incubate at 65°C for 15 min. Then add 17 μl 1 M HEPES, pH 7.5. Samples can be snap-frozen after this step. Isolation of Nascent Transcripts
46. Separate beads using a magnetic stand and transfer samples into YM-10 Amicon columns. Add 500 μl nuclease-free water and centrifuge at 14,000 × g for 10 min at room temperature.
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47. Add another 500 μl nuclease-free water to the columns and centrifuge at 14,000 × g for 12 min. 48. Place columns in a clean tube, invert, and spin at 10,000 × g for 1 min to collect samples. The collected sample volume should be 30 μl.
49. Use isolated cDNA from step 48 to detect gene of interest with PCR/qPCR.
Strand-specific detection of nascent RNA enrichment genome wide Steps 50 to 56 provide a genome-wide alternative to the gene-type-specific method for detection of nascent transcripts. 50. Remove ribosomal RNA (rRNA) with Ribo-Zero Gold Magnetic kit following the kit manufacturer’s instructions. rRNA removal step must be done before you perform the click reaction!
51. Clean up and concentrate rRNA-depleted samples with RNA Clean & Concentrator kit, according to the manufacturer’s protocol. Repeat steps 21 through 42. RNA fragmentation: mix 5 μl First Strand buffer with 10 μl rRNA-depleted nascent RNA, enriched through biotin-streptavidin interaction (from step 42). Incubate at 94°C for 5 min in a thermocycler, then quickly chill samples on ice. 52. For first strand synthesis, add the following to the fragmented RNA: 1.5 μl 100 mM DTT (SuperScript III, Life Technologies) 1.25 μl Random Primers (Life Technologies, cat. no. 48190011) Incubate at 65°C for 3 min, chill on ice, then add the following: 1.5 μl 10 mM dNTPs 0.75 μl RNasin 1.5 μl 100 mM DTT 8 μl nuclease-free water 1.5 μl SuperScript III reverse transcriptase (Life Technologies, cat. no. 18080-044) Incubate at 25°C for 10 min, 50°C for 1 hr, 70°C 15 min, 4°C forever in a thermocycler. 53. Purify reaction products using Amicon YM10 columns as described in steps 46 to 48; eluate should be 50 μl. 54. For second strand synthesis, add the following to the samples from previous step: 2 μl 5× First Strand buffer 0.5 μl 100 mM MgCl2 1 μl 100 mM DTT 15 μl 5× Second Strand buffer (Life Technologies, cat. no. 10812-014) 2 μl 10 mM dNTP mix (use dUTP instead of dTTP for strand-specific sequencing) 0.5 μl E. coli DNA ligase (New England BioLabs, cat. no. M205S) 2 μl E. coli DNA polymerase (New England BioLabs, cat. no. M209L) 0.5 μl E. coli RNase H (New England BioLabs, cat. no. M0297S) Incubate at 16°C for 2.5 hr.
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55. Purify reaction products using 1:1.8 reaction volume/Ampure XP beads; add 135 μl Ampure XP beads and mix by pipetting up and down. Place tubes in magnetic stand for 10 min to capture beads. Wash beads with freshly prepared 80% ethanol without disturbing the bead pellet. Do not remove tubes from magnetic stand.
Wait 2 min and aspirate ethanol. Repeat the wash step. Air dry pellet for 5 to 6 min. It is important not to over dry beads as it lowers elution efficiency.
Resuspend beads in 36 μl nuclease-free water, pipet up and down, and incubate at room temperature for 5 min. Place tubes in magnetic stand for 5 min to capture beads. Transfer eluted DNA to a new tube. 56. Continue with sequencing library construction as described earlier (Bowman et al., 2013; UNIT 4.21, Podnar et al., 2014). 57. UDG treatment for strand specificity: add 2 μl UDG to 30 μl eluted DNA and incubate at 37°C for 30 min. This step should be done just before sequencing library amplification PCR, right after the adapter ligation step. SUPPORT PROTOCOL
ASSESSING CLICK EFFICIENCY WITH RNA DOT-BLOT RNA dot/slot blotting is a simple technique that allows hybridization and analysis of unfractionated RNA. It is similar to DNA dot blotting except for the denaturation step of samples prior to their immobilization on the membrane. The protocol described here is modified from Dolken et al., 2008 (also see UNIT 4.9; Brown et al., 2004).
Additional Materials (also see Basic Protocol) Formamide (Sigma-Aldrich, cat. no. F9037) 20× SSC (Ambion, cat. no. AM9763) Pierce High Sensitivity Streptavidin-HRP (Life Technologies, cat. no. 21134) Nucleic Acid Detection blocking buffer (Thermo Scientific, cat. no. 89880 A) Binding solution (see recipe) Streptavidin-HRP wash buffer (see recipe) Zeta-Probe membrane (Bio-Rad, cat. no. 162-0153) Diethyl pyrocarbonate (DEPC)-treated water (see recipe) Tris-buffered saline and Tween 20 (TBST) Nuclease-free BSA (Sigma-Aldrich, cat. no. B2518) Whatman 3 MM paper Bio-Dot apparatus (Bio-Rad) UV crosslinker (UV Stratalinker 2400) Prepare membrane and assemble manifold for transfer 1. Clean dot-blot manifold with RNaseZap solution and rinse well with DEPC-treated water. 2. Cut membrane to the same size as the manifold and pre-wet membrane in a clean glass tray with 10× SSC for 5 to 10 min. Handle membrane only with RNaseZap-cleaned, blunt-ended forceps. Avoid touching membrane even with gloves on. Isolation of Nascent Transcripts
3. Assemble the membrane and the manifold according to the manufacturer’s instructions. Mark membrane for orientation.
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4. Fill each well with 500 μl nuclease-free water. Check wells for even suction and make sure there are no air bubbles in the wells or air leaks in the manifold assembly. Be careful with the amount of vacuum applied because too much suction can damage the membrane. Use manifold suction setting 3.
Denature RNA samples and immobilize them on the membrane 5. Denature biotinylated RNA at 65°C for 10 min, then chill sample on ice. 6. Add 20 μl 10× binding solution to the denatured and cooled samples. Bring volume up to 200 μl with nuclease-free water. Mix well and transfer mixture to the wells. Fill empty wells with same amount of 2× SSC, and then apply gentle suction. 7. Wash wells with 500 μl 1× binding solution buffer and apply suction. 8. Disassemble manifold; air dry the membrane on clean Whatman 3 MM paper. 9. UV crosslink membrane in UV Stratalinker 2400 using the auto-crosslink setting.
Detect streptavidin-HRP 10. Block membrane with 12 ml Nucleic Acid Detection blocking buffer for 30 min at room temperature on an orbital shaker. 11. Dilute Pierce High-Sensitivity Streptavidin-HRP 1:100 with 12 ml 5% nuclease-free BSA in TBST and incubate 15 min at room temperature on an orbital shaker. 12. Wash membrane once with 1:10 dilution of Nucleic Acid Detection blocking buffer/water 5 min at room temperature. 13. Wash twice with streptavidin-HRP wash buffer 10 min at room temperature. 14. Wash once with TBST 5 min at room temperature. 15. Detect biotin-streptavidin-HRP complex with enhanced chemiluminescence.
REAGENTS AND SOLUTIONS Use nuclease-free water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2.
Binding solution, 10× 100 mM NaOH 10 mM EDTA Store solution up to one year at −20°C Diethyl pyrocarbonate (DEPC)-treated water Add 0.1% diethyl pyrocarbonate to water and mix well. Incubate at 37°C for 2 hr. Autoclave solution to destroy diethyl pyrocarbonate as it may modify RNA if present in the solution. Store at room temperature indefinitely. Mimosine stock solution Dissolve 79 mg mimosine in 6 ml 0.1 N sodium hydroxide (cell culture tested). After mimosine has completely dissolved, add 4 ml DMEM/F-12 medium. Filter and sterilize solution. Aliquot and store at −80°C indefinitely RPE1 medium 500 ml Advanced DMEM/F-12 (Life Technologies, cat. no. 12634-010) 50 ml FBS continued
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5 ml Glutamax (Life Technologies, cat. no. 35050-061) 5 ml penicillin/streptomycin (Life Technologies, cat. no. 15140-122) 17.3 ml 7.5% sodium bicarbonate (Sigma-Aldrich, cat. no. S5761) Mix well, filter, and sterilize Store solution at 4°C for up to 3 months Streptavidin-HRP wash buffer 10× stock solution contains: 100 mM Tris·Cl, pH 7.6 (APPENDIX 2) 100 mM NaCl 10 mM EDTA Add 0.05% Tween 20 fresh to 1× solution each time Store 10× stock solution at room temperature up to 6 months TNE bead-blocking buffer 10× stock solution contains: 100 mM Tris·Cl, pH 7.5 (APPENDIX 2) 10 mM EDTA, pH 8.0 2 M NaCl 20 mg/ml nuclease-free BSA 10 μg/ml poly(deoxyinosinic-deoxycytidylic) acid, sodium salt (polydI-dC) Aliquot and store up to a year at −20°C TNE0.2 10 mM Tris·Cl, pH 7.4 (APPENDIX 2); 1 mM EDTA, pH 8.0; and 200 mM NaCl in nuclease-free water TNE2.0 10 mM Tris·Cl, pH 7.4 (APPENDIX 2); 1 mM EDTA, pH 8.0; and 2 M NaCl in nuclease-free water Store up to a year at room temperature Wash buffer 65 100 mM Tris·Cl, pH 7.4 (APPENDIX 2) 10 mM EDTA 1 M NaCl 0.1% Tween 20 Mix first three items. Store buffer up to a year at room temperature. Add 0.1% Tween 20 fresh each time just before use. COMMENTARY Background Information
Isolation of Nascent Transcripts
Copper-catalyzed azide-alkyne cycloaddition (CuAAC), also known as a “click” reaction (Kolb et al., 2001; Rostovtsev et al., 2002) enables efficient conjugation of azidecontaining molecules to ethynyl-labeled nucleic acids both in vitro (Paredes and Das, 2010) and in vivo (Salic and Mitchison, 2008). These conjugated molecules can then be selectively enriched via affinity purification through biotin-streptavidin interaction. In vivo incorporation of the EU into the nascent transcripts and their microscopic detection via coppercatalyzed cycloaddition (click reaction) of flu-
orescent azides have been described (Jao and Salic, 2008). Standard gene expression analyses on total cellular RNA are only capable of profiling steady-state levels of RNA at a given time, but lack the ability to measure true transcriptional response. In addition, the results are obscured by different RNA decay rates, and are especially biased against transcripts with long half-lives (Friedel and D¨olken, 2009). To circumvent some of these limitations, earlier RNA-decay studies used drugs to inhibit transcription (Bernstein et al., 2002; Raghavan et al., 2002) and utilized nuclear run-on
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assays to detect nascent transcripts in a genespecific manner (Hirayoshi and Lis, 1999) or a genome-wide manner with radiolabeled nucleotides (Garcıa-Martınez et al., 2004) or the nucleotide analog 5-bromouridine 5 triphosphate (BrUTP; Core et al., 2008). These approaches are both technically demanding and, more importantly, experimentally biased as they are cell invasive. In vivo labeling with 5 -bromouridine (BrU) and isolation of nascent transcripts with anti-BrU antibody has also been tested (Ohtsu et al., 2008). While this approach is technically less demanding compared to nuclear run-on-based assays, it is disadvantageous due to BrU toxicity and the relative inefficiency of capturing RNAs with low uridine content. Thiol-containing nucleosides are also known to incorporate into nascent transcripts. While some versions of thiol-modified nucleoside analogs show toxicity, 4-thiouridine (4sU) has been shown to be devoid of cytotoxic effects (Dolken et al., 2008). It was used to profile genome-wide mRNA synthesis and decay rates in yeast (Miller et al., 2011) and mammals (Rabani et al., 2011). Hence, in many aspects, metabolic labeling of nascent RNA with 4sU and EU is similar except for the biotinylation step (Zeiner et al., 2008). One advantage of biotinylation with click chemistry is that it requires much less material in terms of both RNA and biotin. Also, it is a much more rapid reaction than chemical biotinylation of 4sU (30 min versus 3 hr). In addition, unlike 4sU, EU-click chemistry has the potential to be used in whole cells to isolate nascent RNA-associated factors.
Critical Parameters RNA quality High quality RNA is essential for the success of each and every step of this protocol. In particular, RNA integrity should be checked after the copper-catalyzed azide-alkyne cycloaddition step. A good check is to run two reactions for each sample where one reaction is run without copper as a control, to check ligand efficiency in stabilizing copper and to assess copper-related degradation. Pre-mixing ligand (PMDTA or THPTA) and copper (CuSO4 ), then adding the combination to the reaction is beneficial to further avoid copper-catalyzed oxidation of RNA. Another way of reducing the risk of coppermediated RNA oxidation is to degas the reaction by bubbling argon or nitrogen gas.
Click reaction efficiency The basic CuAAC reaction requires copper ions in the +1 oxidation state. Instead of using Cu(I) directly, Cu(II) salts reduced to Cu(I) by ascorbic acid during the reaction are preferred to avoid undesired by-products (Rostovtsev et al., 2002). Sodium ascorbate not only allows a safer reactive copper (i.e., allows a more controlled reaction environment with less oxidative stress) but also extends the half-life of the reactive Cu(I) species in the reaction. It is crucial to maintain high Cu(I) levels at all times during the click reaction for high yield. Using at least a five-fold excess of ascorbate over Cu(II) salt is important for reaction efficiency. Also, sodium ascorbate needs to be prepared fresh. Using a co-solvent such as acetonitrile is important for both copper coordination and RNA integrity. Including a ligand such as PMDTA in the reaction is particularly important as it significantly accelerate the reaction. Another factor that contributes to the reaction rate is the reaction temperature as the click reaction occurs more efficiently at higher temperatures (Meldal and Tornøe, 2008). Control samples Including reactions lacking copper or without EU labeling act as good controls, to allow control of RNA integrity and also to assess pull-down specificity. Next-generation RNA sequencing If nascent RNA will be analyzed with highthroughput sequencing then samples should be depleted of rRNA before biotinylation with the click reaction because rRNA-depletion kits utilize streptavidin-coated beads.
Troubleshooting The RNA yield with ethanol precipitation after the click reaction (Basic Protocol, step 28) gives the first indication of RNA integrity. Unless there is RNA degradation there should not be any difference in the amount of starting material and precipitated sample. More reliable assessment of RNA integrity is provided by comparing the click reaction sample and its control reaction sample (which does not contain CuSO4 ) with RT-PCR for a known expressed target. For example, less PCR product from the click reaction than from the (control) reaction in the absence of copper indicates a failure in copper stabilization and/or an oxidized reaction environment. Use of freshly
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prepared PMDTA and fresh sodium ascorbate is recommended. Also, increasing the time argon is bubbled through the reaction to degas the mixture can be beneficial. After the streptavidin pull-down step, control samples (i.e, samples from reactions run in the absence of CuSO4 or EU) should not produce any detectable PCR product. Any PCR products from those samples suggest a nonspecific pull down (false positive). The bead-blocking step is crucial to avoid nonspecific pull down. The bead-blocking step and/or 65°C wash step can be extended to avoid this result.
Anticipated Results This protocol allows isolation of nascent transcripts using the desired conditions depending on the experimental design. RT-qPCR after nascent RNA pull down can be used to examine whether a gene of interest is newly synthesized. Alternatively, nextgeneration sequencing after nascent transcript isolation will provide a genome-wide map of newly synthesized RNAs. When labeling cellcycle-specific nascent transcripts, replicationdependent canonical histones serve as a useful positive control for S phase and can be used as a negative control for M phase. Another control is the Xist transcript as it is maintained at the same levels throughout the cell cycle.
Time Considerations Cell cycle synchronization, EU labeling and sample collection can be completed in 2 days. Total RNA isolation from those samples and DNase treatment takes about 2 hr. If pulled down RNA will be analyzed with highthroughput sequencing then samples should be depleted of rRNA. This additional step takes about an hour. Biotinylation with the click reaction, followed by sample precipitation and streptavidin pull down of nascent transcripts, requires 4 to 5 hr. The cDNA synthesis step should be performed immediately once nascent RNAs are pulled down, which takes about 2 hr, then the RNA of interest can be searched in the nascent RNA pull-down material using a regular PCR reaction in about 1 hr.
Acknowledgements Isolation of Nascent Transcripts
Ozlem Yildirim is an HHMI Fellow of The Damon Runyon Cancer Research Foundation (DRG-2156-13). The author would like to acknowledge Kingston Lab members for stimulating discussions.
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