Nucleotide-Level Profiling of m5C RNA Methylation - Springer

Chapter 16 Nucleotide-Level Profiling of m5C RNA Methylation Tennille Sibbritt, Andrew Shafik, Susan J. Clark, and Thomas Preiss Abstract Mapping the position and quantifying the level of 5-methylcytosine (m5C) as a modification in different types of cellular RNA is an important objective in the emerging field of epitranscriptomics. Bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification and detection of candidate sites by sequencing on the Illumina MiSeq platform. Key words 5-Methylcytosine, Epitranscriptomics, Bisulfite conversion, Next-generation sequencing, MiSeq

1  Introduction Cellular RNAs can be richly modified with more than one hundred known, chemically and structurally distinct nucleoside modifications [1–3]. The emerging field of epitranscriptomics [4–6] is enabled by the development of high-throughput mapping methods for RNA modifications, typically based on a next-generation sequencing (NGS) readout. Transcriptome-wide positions of 5-methylcytosine (m5C) [7], N6-methyladenosine [8–10], and pseudouridine [11–13] have each been reported in this way. To detect m5C in RNA, a range of methods have been developed, including the direct (meRIP [14]) or indirect (aza-IP [15], miCLIP [16]) immunoprecipitation of methylated RNA. Of particular interest here, the bisulfite conversion approach in popular use for DNA methylation detection has also been adapted to RNA [7, 17–19]. Bisulfite conversion of nucleic acids takes advantage of the differential chemical reactivity of m5C compared to unmethylated cytosines; unmethylated cytosines are deaminated to uracil while m5C remains as a cytosine. We recently adapted an RNA bisulfite conversion method [17] for a NGS-based transcriptome-wide readout and mapped thousands of novel candidate m5C sites in a variety of RNA biotypes, Erik Dassi (ed.), Post-Transcriptional Gene Regulation, Methods in Molecular Biology, vol. 1358, DOI 10.1007/978-1-4939-3067-8_16, © Springer Science+Business Media New York 2016

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including mRNA [7]. Here, we detail our protocols for RNA bisulfite conversion and locus-specific, semiquantitative PCR-­ based detection of non-converted sites. Sequencing of PCR amplicons is conveniently done on the Illumina MiSeq platform, as this affords multiplexing of multiple distinct amplicons while still achieving ample read depth for estimating the proportion of m5C at targeted positions. For instance, each of the 24 indexed adaptors from the TruSeq DNA LT Sample Prep Kit could be assigned to a separate cellular RNA source material, and multiple RNA loci/PCR amplicons per sample could be included in the sequencing library, potentially generating hundreds of independent quantitative measurements of the m5C level in a single MiSeq run (Fig. 1).

2  Materials Prepare all solutions using DNase- and RNase-free H2O and analytical grade reagents. Prepare and store all reagents at room temperature unless indicated otherwise. Diligently follow all safety and waste disposal regulations when performing experiments. Prepare and perform bisulfite conversion, cDNA synthesis, and PCR amplification experiments in a PCR and plasmid-free area. 2.1  In Vitro Transcription Components

1. pRL Renilla Luciferase Reporter Vectors (pRL-TK) (Promega). 2. MEGAScript® T7 Kit (Life Technologies). 3. TURBO™ DNase (Life Technologies). 4. Phase Lock Gel Heavy (1.5 mL) (5 Prime). 5. UltraPure™ Phenol:Water (3.75:1 v/v) (Life Technologies). 6. Chloroform. 7. Glycogen (5 mg/mL) (Life Technologies). 8. Agilent RNA 6000 Nano Kit (Agilent).

2.2  Sodium Bisulfite Conversion Components

1. Sodium bisulfite solution: 40 % (w/v) sodium metabisulfite, 0.6 mM hydroquinone, pH 5.1. 0.6 M Hydroquinone: Weigh 66 mg hydroquinone and place into a 1.5 mL tube. Add H2O to 1 mL and cover in foil to protect from light. Place in an orbital shaker to dissolve (see Note 1). 40 % (w/v) sodium bisulfite: Dissolve 8 g sodium metabisulfite in 20 mL H2O in a 50 mL falcon tube and vortex until it completely dissolves. Add 20 μL 0.6 M hydroquinone to the 40 % sodium bisulfite solution, vortex, and adjust pH to 5.1 with 10 M NaOH (see Note 2). Filter the solution through a 0.2 μm filter. Cover in foil to protect from light.

Nucleotide-Level Profiling of m5C RNA Methylation Sample 2

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Bisulfite conversion Reverse transcription PCR amplification in triplicate Amplicon A Amplicon B

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Amplicon A Amplicon B

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Pooling of triplicate amplicons

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A Purification, quantification & pooling of multiple amplicons for each sample

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MiSeq library preparation (End repair, A-tailing, ligation of indexed adaptors, PCR enrichment)

Pooling of samples and MiSeq run

Fig. 1 Protocol overview, showing workflow and pooling strategy for effective sequencing. Total RNA spiked with the R-Luc in vitro transcript is bisulfite converted. The bisulfite-converted RNA is reverse transcribed and candidates of interest, as well as the positive (tRNAAsp(GUC) and tRNALeu(CAA)) and negative (Renilla Luciferase in vitro transcript) controls, are PCR amplified in triplicate for each sample to minimize PCR amplification bias. The triplicate amplicons are then pooled for each sample and subjected to purification. The purified amplicons are quantified and an equal amount of each amplicon is pooled for each sample. Following this, library preparation is performed using the TruSeq DNA LT Sample Prep Kit, which involves end repair and A-tailing of the amplicons, ligation of the indexed adaptors to the amplicons to enable multiplexing of samples, and enrichment of the libraries by PCR. Each library is then pooled, a PhiX control library is spiked in, and the libraries are subjected to sequencing on the MiSeq platform

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2. 1 M Tris–HCl, pH 9.0. 3. Micro Bio-Spin® P-6 Gel Columns, Tris buffer (Bio-Rad). 4. Mineral oil. 5. 100 % ethanol. 6. 75 % ethanol. 7. 3 M sodium acetate, pH 5.2. 8. 5 mg/mL glycogen (Life Technologies). 2.3  cDNA Synthesis Components

1. SuperScript III® Reverse Transcriptase Kit (Life Technologies). 2. 10 mM mixed dNTPs. 3. 20× random primer mix: 35 μM hexamers, 25 μM T12VN. Oligo sequence for hexamers: NNNNNN Oligonucleotide sequence for T12VN: NVTTTTTTTTTTTT

2.4  PCR Amplification Components 2.5  Agarose Gel Electrophoresis and PCR Purification Components

1. Platinum® Taq DNA Polymerase Kit (Life Technologies). 2. 10 mM mixed dNTPs. 1. Seakem® LE Agarose (Lonza). 2. EZ-vision® Three DNA Dye and Buffer 6× (Amresco). 3. 1 Kb Plus DNA ladder (Life Technologies). 4. 1× TAE buffer: Make up 50× TAE buffer by combining 424 g Tris base, 57.1 mL acetic acid, and 100 mL 0.5 M EDTA (pH 8.0) and make up to 1 L in H2O. To make 1× TAE buffer, combine 40 mL 50× TAE buffer with 1.96 L H2O. 5. Wizard SV Gel and PCR Clean-Up System (Promega).

2.6  Library Preparation Components

1. TruSeq DNA LT Sample Prep Kit v1/v2 (Illumina). 2. MinElute PCR Purification Kit (Qiagen). 3. Agencourt AMPure XP beads (Beckman Coulter). 4. Tween 20 (Sigma). 5. EBT buffer: 10 mM Tris–HCl (pH 8.5) and 0.1 % Tween 20. Add 19.8 mL H2O to 0.2 mL 1 M Tris–HCl (pH 8.5), and then add 20 μL Tween 20 to the solution (see Note 3). Vortex solution thoroughly to ensure that Tween 20 is mixed throughout the solution. 6. Library dilution buffer: 10 mM Tris–HCl (pH 8.0) and 0.05 % Tween 20. Add 19.8 mL H2O to 0.2 mL 1 M Tris–HCl (pH 8.0) followed by 10 μL Tween 20. Vortex solution thoroughly to ensure that Tween 20 is mixed throughout the solution. 7. Library Quantification Kit-Illumina/Universal (Kapa Biosystems). 8. 0.2 M NaOH. 9. MiSeq Reagent Kit v2 (300 cycles) (Illumina) (see Note 4).

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3  Methods Carry out all procedures at room temperature unless otherwise specified. 3.1  RNA Extraction and DNase Treatment

3.2  Generation of the Renilla Luciferase (R-Luc) In Vitro Transcript Spike-In Control

Total cellular RNA is extracted directly from adherent cells with 1 mL of Tri Reagent® as per the manufacturer’s protocol. RNA is then treated with TURBO™ DNase as per the manufacturer’s protocol. 1. Linearise the pRL-TK vector using BamHI according to the MEGAScript® T7 Kit protocol. 2. Perform in vitro transcription according to the MEGAScript® T7 Kit protocol using 1 μg linearized DNA. An incubation period of 4 h at 37 °C with the kit components is sufficient. 3. Add 2 U TURBO™ DNase and incubate at 37 °C for 30 min. 4. Transfer the reaction to a Phase Lock Gel Heavy (1.5 mL) tube and make the volume of the reaction up to 100 μL with H2O. 5. Add an equal volume of UltraPure™ Phenol:Water (3.75:1 v/v) and chloroform, shake vigorously for 15 s, and centrifuge at 16,000 × g for 5 min. 6. Add the same volume of chloroform as step 5 to the tube, shake vigorously for 15 s, and centrifuge at 16,000 × g for 5 min again. 7. Transfer the aqueous phase to a clean 1.5 mL tube. Add 1/10 volume 3 M sodium acetate, 3 volumes of 100 % ethanol, and 1 μL glycogen (5 mg/mL), vortex, and precipitate the RNA overnight at −80 °C. 8. Centrifuge RNA at 17,000 × g at 4 °C for 30 min and carefully remove the supernatant. 9. Add 1 mL 75 % ethanol to the RNA, invert ~5 times and centrifuge at 7500 × g at 4 °C for 5 min (see Note 5). 10. Carefully remove the supernatant and let the pellet air-dry for 10–15 min (see Note 6). 11. Resuspend the RNA in H2O. 12. Treat 10  μg in vitro transcript with 2 U TURBO™ DNase according to the manufacturer’s protocol at 37 °C for 30 min to remove any residual template DNA. 13. Assess the size and integrity of the in vitro transcript using a RNA 6000 Nano Chip on the Agilent® 2100 Bioanalyzer according to the manufacturer’s protocol.

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3.3  Bisulfite Conversion of RNA

1. Add 1/1000 R-Luc in vitro transcript to 2–4 μg DNasetreated RNA (see Note 7). The combined volume of the RNA sample and in vitro transcript should be 55 μL, concentrate it down to ~55 μL or less using a vacuum concentrator and make the volume up to 55 μL in H2O. If the volume is 2; however this does not hinder subsequent reactions. 11. ~500 ng is lost during this procedure, and we find that 13–15 μL of H2O per 2 μg RNA used in the bisulfite conversion reaction results in concentrations of ~100 ng/μL. 12. For transcriptome-wide detection of m5C (bsRNA-seq), confirmation of fragment size using the Agilent® 2100 Bioanalyzer RNA 6000 Nano Chip according to the manufacturer’s protocol is required. This is not required for preparation of RNA for locus-specific sequencing using Sanger sequencing or the MiSeq platform. 13. Inefficient bisulfite conversion may result in unconverted cytosines, so it is necessary to ensure the primers are not biasing towards converted cytosines.

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14. tRNAAsp(GUC) is known to contain m5C sites at C38, C48, and C49, and tRNALeu(CAA) is known to contain a m5C site at C34. We have previously used these transcripts as positive controls for m5C sites. 15. Longer amplicons increases the propensity of detecting non-­ converted cytosines in RNA exhibiting strong secondary structure. We have previously experienced this for amplicons derived from rRNA. 16. A “touchdown” PCR is performed to increase the specificity of the product. The first phase uses a higher annealing temperature, amplifying the specific product. The annealing temperature at each subsequent cycle is decreased by 1 °C to approximately 5 °C below the lowest primer melting temperature. In the second phase, a standard PCR protocol is implemented using the lowest annealing temperature used in the first phase. 17. Use an extension time of 30 s for amplicons of ~200 bp. For amplicons