Methods in Molecular Biology DOI 10.1007/7651_2014_192 © Springer Science+Business Media New York 2014

RNA-Sequencing of Staphylococcus aureus Messenger RNA Ronan K. Carroll, Andy Weiss, and Lindsey N. Shaw Abstract RNA-sequencing (RNA-seq) is a technique that employs next-generation DNA-sequencing technology to simultaneously sequence all of the RNA transcripts in a cell. It can provide valuable insights into transcript and operon structure, and is rapidly replacing expression microarrays as the technique of choice for determining global gene expression profiles in bacteria. Herein we outline the procedures involved in performing RNA-seq with samples of RNA from Staphylococcus aureus. We draw particular attention to key aspects of sample preparation, such as RNA integrity and removal of ribosomal RNA, and provide details of critical steps in downstream data analysis. Keywords: RNA-seq, RNA-sequencing, RPKM, rRNA reduction, RNA enrichment, S. aureus

1

Introduction The introduction of next-generation DNA sequencing (NGS) has facilitated the development of techniques and applications for highthroughput genetic analysis that were previously not possible. Perhaps the most beneficial application of NGS in microbial research has been the introduction of RNA-sequencing (RNA-seq). In this technique total RNA from the bacterial cell is purified and sequenced using NGS technology. The resulting reads, once aligned to a reference genome, can be used to quantitate gene expression, determine transcript and operon structure, and identify noncoding RNA species, in essence giving a “snapshot” of global RNA expression within the cell. An overview of the RNA-seq experimental procedure is presented in Fig. 1. RNA samples are isolated and contaminating genomic DNA removed using an RNase-free DNase (Fig. 1a–b). RNA samples are then enriched, a process whereby ribosomal RNA (rRNA) is removed (Fig. 1b–c). Following enrichment the RNA-sequencing process begins. Each NGS platform has specific RNA-seq protocols that are used to process enriched RNA samples. In most cases the enriched RNA is fragmented before being reverse-transcribed into cDNA and sequenced (Fig. 1d). The sequencing reads are then aligned to a reference genome and analyzed to determine gene expression values (Fig. 1e).

Ronan K. Carroll et al.

Fig. 1 Schematic overview of RNA-sequencing. (a) Total RNA purified from S. aureus cells contains messenger RNA (mRNA), ribosomal RNA (rRNA) as well as small amounts of genomic DNA that are co-purified with the RNA.

RNA-seq in S. aureus

Each NGS platform has its own set of unique protocols for processing enriched RNA. Platform-specific, detailed protocols (as well as instructional videos) are freely available online, and therefore, there is little value in reiterating them in detail herein. In addition, many of these procedures are now available as services at core facilities, removing the need for individual labs to perform these steps themselves. As such, it is our intention to focus this protocol on critical aspects of sample preparation that must be carefully performed to generate samples of sufficient quality for RNA-seq. We provide detailed protocols for RNA isolation, DNase treatment, enrichment, and quality evaluation. In addition we include important points for RNA-seq data handling which facilitate accurate downstream analysis.

2 2.1

Materials RNA Isolation

1. RNeasy Mini Kit (Qiagen), Buffer RLT must be prepared fresh each day. Prepare a sufficient quantity of buffer RLT by adding 10 μl of β-mercaptoethanol to 1 ml buffer RLT provided in the Qiagen RNeasy kit. 2. Nuclease-free 1.5 ml tubes. 3. TE buffer: 10 mM Tris pH 8.0, 1 mM EDTA. 4. Lysing matrix B 2 ml tubes (MP Biomedical). 5. Mini-beadbeater. 6. Ethanol (100 %). 7. Nuclease-free water. 8. RNaseZap (Life Technologies).

2.2 DNase Treatment of Purified RNA

1. Turbo DNA-free kit (Ambion). 2. Agilent 2100 Bioanalyzer or 2200 TapeStation. 3. Agilent RNA 6000 Nano kit.

2.3

RNA Enrichment

1. ERCC RNA spike in mix (Life Technologies). 2. Ribo-Zero Magnetic (Epicentre).

Kit

for

Gram-Positive

Bacteria

ä Fig. 1 (continued) (b) Contaminating DNA is removed using an RNase-free DNase and the integrity of the purified RNA samples determined by bioanalyzer. Samples with a RIN >9.7 are suitable for RNA-seq. (c) The RNA enrichment procedure removes rRNA leaving mRNA transcripts. (d) The mRNA is fragmented, reversetranscribed into cDNA, and sequenced using next-generation DNA-sequencing technology. (e) Sequencing reads corresponding to rRNA are filtered out and the remaining reads are aligned to a reference genome. Expression values for each gene are calculated based upon the number of reads aligning to that gene

Ronan K. Carroll et al.

3. MicrobExpress Technologies).

Bacterial mRNA Enrichment

Kit

(Life

4. Magnetic rack for 1.5 ml tubes. 5. 0.2 ml tubes. 6. Thermocycler. 7. Ethanol (70 %). 8. Agilent RNA 6000 Pico kit. 2.4

RNA-Sequencing

1. Ion Total RNA-Seq Kit v2. 2. Ion PGM™ Template 200 Kit. 3. Ion 318™ Chip. 4. Ion PGM™ 200 Sequencing Kit. 5. Ion Xpress™ RNA-Seq Barcode Kit (optional). 6. IonTorrent PGM. 7. IonTorrent OneTouch.

2.5

3 3.1

Data Analysis

1. CLC Genomics Workbench software.

Methods RNA Isolation

While a variety of RNA isolation procedures are available, the protocol below is based upon the Qiagen RNeasy kit. When isolating RNA, use dedicated RNase-free reagents throughout (see Note 1). 1. Grow bacterial cultures as required and pellet corresponding to 1  109 CFU (see Notes 2 and 3).

cells

2. Immediately freeze pellets and store at 80  C prior to RNA isolation (see Note 4). 3. Thaw pellets and resuspend in 100 μl TE buffer pH 8.0. 4. Transfer resuspended cells to a Lysing matrix B tube and bead beat for 60 s. 5. Add 650 μl of buffer RLT containing β-mercaptoethanol to each tube and repeat the bead beating procedure for an additional 60 s. 6. Centrifuge samples at full speed in a microcentrifuge for 1 min. 7. Withdraw 600 μl of supernatant from each tube, being careful not to disturb the pelleted beads/cellular debris, and mix with 900 μl of 100 % ethanol in a 1.5 ml tube (see Note 5). 8. Immediately transfer 600 μl of the lysate–ethanol mix to an RNeasy mini spin column.

RNA-seq in S. aureus

9. Centrifuge for 30 s at maximum speed, discard the flowthrough, reconnect the RNeasy mini spin column to the collection tube and transfer another 600 μl of the lysate–ethanol mix to the RNeasy mini spin column. 10. Repeat step 9, transferring the remainder of the lysate–ethanol mix to the RNeasy mini spin column, and centrifuge. 11. Wash the column with 700 μl of buffer RW1. 12. Transfer the spin column to a new 2 ml collection tube. 13. Wash the column twice with 500 μl of buffer RPE (see Note 6) discarding the flow-through each time. 14. Reconnect the spin column and the 2 ml collection tube and centrifuge at full speed for 2 min. 15. Place the spin column in a clean 1.5 ml tube and elute the RNA by adding 53 μl of nuclease-free water to the column, wait 1 min, then centrifuge at full speed for 1 min. 3.2 DNase Treatment of Purified RNA

To ensure complete removal of contaminating DNA, treat the isolated RNA samples with DNase I (see Note 7) 1. Set up DNase treatment reactions by adding 6 μl of 10 DNase buffer and 1 μl of DNase I to the 53 μl of RNA eluted in step 15 (above). 2. Incubate at 37  C for 60 min. 3. Stop the DNase I reaction by adding 7 μl of DNase deactivation buffer, mix by pipetting, and centrifuge samples at 10,000  g for 2.5 min. 4. Carefully remove 42 μl of the supernatant to a clean tube (taking care not to disturb the white pelleted material), and assess the quality and quantity of RNA using an Agilent bioanalyzer. 5. Store RNA samples at 80  C.

3.3

RNA Enrichment

RNA samples for RNA-seq experiments must be of the highest quality. Any degradation of the ribosomal RNA will result in poor RNA enrichment and subsequently increase the number of rRNA reads in the RNA-seq data set. To ensure RNA samples are of the highest quality they must be assessed on an Agilent bioanalyzer instrument using an RNA 6000 nano chip. 1. Following the manufacturer’s protocol assess the quality and quantity of RNA samples by running 1 μl of each on an Agilent RNA 6000 nano chip. RNA samples with an RNA integrity number (RIN) greater than or equal to 9.8 are of a suitable quality for use in RNA-seq (see Note 8).

Ronan K. Carroll et al.

2. Assess the quality of the RNA samples and proceed to the enrichment step only with those where the RIN > 9.7. 3. Using quantification from the bioanalyzer, determine the volume of each RNA sample that equals 4 μg RNA and add nuclease-free water to bring the total volume to 24 μl (see Note 9). 4. Add 2 μl of a 1/100 dilution of ERCC RNA spike-in-mix to each sample (see Note 10). 5. Proceed to rRNA removal. Efficient removal of rRNA is essential prior to performing RNA-seq reactions. The method below combines the use of two commercially available rRNA removal kits. The Ribo-Zero Magnetic Kit for Gram-Positive Bacteria is used first, followed by a second round of purification using a MicrobExpress Bacterial mRNA Enrichment Kit (see Note 11). S. aureus RNA samples processed according to this protocol routinely generate RNA-seq data sets where total rRNA reads

RNA-Sequencing of Staphylococcus aureus Messenger RNA.

RNA-sequencing (RNA-seq) is a technique that employs next-generation DNA-sequencing technology to simultaneously sequence all of the RNA transcripts i...
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