RNA 5-Methylcytosine (m5C) Sequencing by Illumina Protocol

RNA Extraction, Purification, and DNase Treatment

Total RNA is extracted and purified directly from tissue with 1 mL of TRIzol™ as per the manufacturer's protocol. RNA is then treated with TURBO™ DNase as per the manufacturer's protocol. Assess the integrity of the RNA by using a RNA 6000 Nano Chip on the Agilent 2100 Bioanalyzer according to the manufacturer's protocol.

Generation of the MGFP In Vitro Transcript Spike-in Control

1. Linearize the phMGFP vector by using the restriction enzyme XbaI and purify the linearized DNA vector according to the HiScribe T7 In Vitro Transcription Kit protocol.
2. Perform in vitro transcription according to the HiScribe T7 In Vitro Transcription Kit protocol by using 1μg of 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™ tube and make the volume of the reaction up to 100μL with ultrapure H2O.
5. Add an equal volume of phenol:water and chloroform, shake vigorously for 15 s, and centrifuge at 15,000 × g for 5 min.
6. Add the same volume of chloroform as in step 5 to the tube, shake vigorously for 15 s, and centrifuge at 15,000 × 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 1μL glycogen; vortex; and precipitate the RNA overnight at −80 °C.
8. Centrifuge RNA at 17,000 × g at 4 °C for 60 min and carefully remove the supernatant.
9. Add 1 mL 75% ethanol to the RNA, invert five times, and centrifuge at 7500 × g at 4 °C for 10 min.
10. Carefully remove the supernatant and let the pellet air-dry for approximately 15 min.
11. Resuspend the RNA in 25μL of ultrapure H₂O.
12. Optional step: Treat 5μg of in vitro-transcribed MGFP transcript with 2 U TURBO™ DNase according to the manufacturer's protocol at 37 °C for 30 min.
13. Assess the integrity and size of the MGFP in vitro transcripts by using an RNA 6000 Nano Chip on the Agilent 2100 Bioanalyzer according to the manufacturer's protocol.

Bisulfite Conversion of RNA

1. Add 1/2000 of the MGFP RNA transcript to 2μg DNase-treated purified total RNA. Increase the volume of the RNA sample to 20μL with ultrapure H2O.
2. Denature RNA by heating to 75 °C for 5 min in a heat block.
3. Preheat the sodium bisulfite solution to 75 °C, add 100μL to the RNA, vortex thoroughly, and briefly spin in a microcentrifuge (13,000 × g for 1 min).
4. Overlay the reaction mixture with 100μL of mineral oil. Cover the tube in aluminum foil to protect the reaction mixture from light.
5. Incubate at 75 °C for 4 h in a heat block.
6. About 15 min before the bisulfite conversion reaction is complete, prepare two Micro Bio-Spin Columns for each conversion reaction by allowing the Tris solution in the column to drain into a collection tube. Discard the Tris flow-through, place the column back into the collection tube, and centrifuge at 1000 × g for 2 min. Transfer each column to a clean 1.5 mL tube.
7. Remove the bisulfite reaction mixture from the heat block and gently transfer the aqueous layer (that is under the mineral oil) containing the sodium bisulfite/RNA mixture to the Micro Bio-Spin column.
8. Centrifuge at 1000 × g for 4 min.
9. Carefully transfer the eluate into the second Micro Bio-Spin column placed in a 1.5 mL tube and repeat step 8.
10. Preheat the temperature of the heat block to 75 °C in preparation for step 12.
11. Add an equal volume of 1 M Tris–HCl pH 9.0 to the second eluate, vortex, spin briefly, and then overlay with 175μL of mineral oil. Cover the tube in aluminum foil to protect the reaction mixture from light.
12. Incubate at 75 °C for 1 h in the heat block.
13. Transfer the bottom aqueous layer containing the RNA to a clean 1.5 mL tube.
14. Precipitate the bisulfite-treated RNA and resuspend the bisulfite-converted RNA in H₂O.

Bisulfite Oligonucleotide Primer Design for cDNA Synthesis and PCR

1. For efficient parallel amplification of 48 target amplicons on the Fluidigm Access Array, use targeted cDNA synthesis to reduce the amplification of spurious amplicons. Targeted cDNA synthesis is achieved by designing reverse transcriptase (RT) primers 30–40 nt 3' of the cytosine(s) to be assayed.
2. Design primers for the first round of PCR amplification so that small amplicons are 170–200 bp, to allow efficient amplification. As the G/C content in the template is low, design long primers to ensure that a Tm is in the rage of 59–61 °C. Add the CS1 sequence (5'-TACGGTAGCAGAGACTTGGTCT-3') to the forward primer gene-specific sequence (GSS) and CS2 (5'-ACACTGACGACATGGTTCTACA-3') to the reverse primer GSS. For the second PCR amplification, use the forward primer containing the complementary sequences to the P5 Illumina flow cell combined with CS1 (P5_CS1) and the reverse primer containing the barcode, and complementary sequences to the P7 Illumina flow cell combined with CS2 (P7_BC_CS2) primer.

cDNA Synthesis

1. Mix 500 ng of bisulfite-converted RNA, 1μL of 1 mM dNTP mix, and 2μL of 10× pooled primer mix and add ultrapure H₂O to a final volume of 13μL. Incubate the mix at 65 °C for 5 min to denature the RNA.
2. Reverse transcribe the bisulfite-converted RNA using SuperScript™ III Reverse Transcriptase according to the manufacturer's protocol. Add either pooled 48 RT primers for parallel Access Array amplification or random hexamers for single-PCR amplicons.
3. After the reaction is complete, dilute the cDNAs 1:10 in ultrapure H₂O for PCR amplification.

Individual PCR Amplification, Quantification, and Pooling

1. For a 10μL PCR, add 0.2μL of KAPA HiFi DNA Polymerase, 2μL of 5× HiFi Fidelity buffer (with MgCl2), 0.3μL of 10 mM dNTP, 0.4μL of 10μM forward primer (CS1_GSS), 0.4μL of 10μM reverse primer (CS2_GSS), 1μL of diluted cDNA, and H2O to a final volume of 10μL. Perform PCR for each amplicon in triplicate.
2. Gently finger vortex, briefly centrifuge, and place into a preheated thermal cycler.
3. Perform a two-step thermal cycling PCR program.
4. Pool the triplicates and perform an AMPure bead cleanup at a ratio of 1.8:1 to remove unincorporated primers and primer dimers. Repeat this step.
5. Assess PCR amplicon size and concentration after separation on a Shimadzu Microchip Electrophoresis System MCE®-202 MultiNA.
6. Normalize the concentration of each amplicon in the experiment by dilution with H2O to a concentration in the range of 0.5–5 ng/μL.
7. Perform the barcoding and Illumina adapter addition PCR. In a 10μL PCR, add 0.2μL of KAPA HiFi DNA Polymerase, 2μL of 5× HiFi Fidelity buffer (with MgCl2), 0.3μL of 10 mM dNTP, 1μL of 10μM forward primer (P5_CS1), 1μL of 10μM reverse primer (P7_CS2), 2μL of diluted PCR amplicon, and H2O to a final volume of 10μL.
8. Gently finger vortex, briefly centrifuge, and place into a preheated thermal cycler.
9. Perform a two-step thermal cycling PCR program.
10. Assess PCR amplicon size and concentration after separation on a Shimadzu Microchip Electrophoresis System MCE®-202 MultiNA.
11. Pool the amplicons in equimolar concentration and purify them using AMPure beads according to the manufacturer's protocol. Use a ratio of beads to pooled amplicons of 0.9:1 to ensure binding of amplicons and not primer dimers or unincorporated primers.
12. First estimate the DNA concentration using a Qubit dsDNA Broad Range Assay Kit according to the manufacturer's protocol. Then accurately assess the DNA concentration by using KAPA Library Quantification Kit for Illumina® Platforms. Perform serial dilution of the pooled amplicons such that they fall into the dynamic range of the assay of 5.5–0.000055 pg/μL.

MiSeq Sequencing

1. Prepare the sample sheet using the Illumina Experiment Manager by following the manufacturer's protocol.
2. Dilute the library to 10 nM in EBT buffer based on the concentrations determined by the qPCR. From this point, keep the libraries on ice.
3. Dilute the PhiX control library to 2 nM by adding 8μL EBT buffer to 2μL of the 10 nM PhiX control library.
4. Denature the pooled libraries and PhiX control library separately by adding 10μL of 0.2 M NaOH to 10μL of the 2 nM libraries.
5. Vortex thoroughly to mix and centrifuge at 1000 × g for 30 s. Incubate at room temperature for 5 min.
6. Dilute the denatured pooled libraries and PhiX control library separately to 20 pM by adding 980μL pre-chilled HT1 to 20μL denatured libraries.
7. Dilute the 20 pM pooled libraries and PhiX control library separately to 10 pM by adding 500μL pre-chilled HT1 to 500μL 20 pM libraries.
8. Combine 100μL of the 10 pM PhiX control library with 900μL of the 10 pM pooled libraries and vortex to mix.
9. Load 600μL of the final sample into the cartridge. Ensure that air bubbles are removed by gently tapping the cartridge.
10. Perform the sequencing run according to the manufacturer's protocol.

Reference:

1. Li J, Wu X, Do T, et al. Quantitative and Single-Nucleotide Resolution Profiling of RNA 5-Methylcytosine[M]//RNA Modifications. Humana, New York, NY, 2021: 135-151.