Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate

WO-Seq: a bisulfite-free and base-resolution sequencing method based on peroxotungstate oxidation is presented for the identification of hm5C sites in the transcriptome. Combining the peroxotungstate oxidation with TET enzyme oxidation, m5C can also be detected in a procedure termed TET-Assisted WO-Seq (TAWO-Seq).


Experimental Procedures
Preparation of model RNA oligo 60-mer model RNA and 73-mer model RNA with C, m 5 C or hm 5 C were generated by the HiScribeTM T7 High Yield RNA Synthesis Kit using CTP, 5-Methoxycytidine-5'-Triphosphate or 5-Hydroxymethylcytidine-5'-Triphosphate (Trilink Biotech), along with ATP, GTP and UTP. Synthesized RNA was purified by phenol-chloroform extraction and ethanol precipitation. RNA was then dissolved in 50 L DNase/RNase free water (Invitrogen) and the concentration and quality were checked using NanoDrop™ 2000/2000c Spectrophotometers (ThermoFisher) and NovexTM 15% TBE-Urea Gel (ThermoFisher). 1 L RiboLock RNase Inhibitor (Thermo) was added to the purified model RNA, and all of the RNA was stored at -80 o C.

Oxidation of model RNA by peroxotungstate
was prepared according to the reported procedures (Chem. Soc. Dalton Trans., 1989, 1203). The synthesized oxidant was stored at room temperature. Generally, 2 g model hm 5 C-containning RNA was incubated in a working solution of 5 mM peroxotungstate, 1 U / L RNase inhibitor, 100 mM sodium chloride in 200 mM sodium phosphate buffer (pH = 7.0) at 60 o C. After 4 hours, model RNA was purified on Zymo-Spin columns (Zymo Research) and eluted in 10 L DNase/RNase free water. Then, the reacted RNA was incubated in the same oxidation condition for another 4 hours, and purified by Micro Bio-Spin 6 column (Bio-Rad). Qubit TM RNA HS Assay Kit (Thermo Fisher) was used to measure the concentration, and the product was stored at -20 o C waiting for further analysis.

Sensitivity and RNA degradation test
RNA samples of different combination of hm 5 C-containing RNA1 and normal rC-containing RNA1 were prepared, which contained 0%, 25%, 50%, 75% and 100% of hm 5 C-containing RNA1. These samples were treated by peroxotungstate oxidation described above, and then analyzed by HPLC-MS.
Total RNA of mESC was extracted using TRIzol TM Reagent (Invitrogen TM ). Same amount of the total RNA sample was treated by peroxotungstate or EZ RNA Methylation TM Kit (Zymo Research). Control untreated RNA and these treated RNA were analyzed by agarose gel.

MALDI-MS analysis and HPLC-MS analysis
Oxidized product of hm 5 C-containing RNA1 was analyzed by MALDI-MS and HPLC-MS/MS. For MALDI-MS, about 200 ng oxidized RNA was digested using 1 L 1000 U / L RNase T1 (Thermo Fisher) at 37 o C for 20 min, and purified by desalting resins. Then, the digested RNA fragments were characterized by Voyager-DE MALDI-TOF (matrix-assisted laser desorption ionization timeof flight) Biospectrometry Wrokstation.
For HPLC-MS/MS, about 50 ng RNA sample was digested using DNA degradase plus TM (1 U / L, Zymo Research) in the presence of 40 nM deaminase inhibitors erythro-9-amino--hexyl--methyl-9H-purine-9-ethanl hydrochloride (Sigma-Aldrich). After incubation at 37 o C for 2 hours, equal volume of solvent A (10 mM ammonium acetate, pH = 6.0) of the HPLC method was added to the solution, and the resulting solution was filtered with Amicon Ultra-0.5 mL 10 K centrifugal filters (Merck Millipore) to remove the proteins.
The HPLC-MS/MS analysis was carried out with 1290 Infinity LC Systems (Agilent) coupled with a 6495B Triple Quadrupole Mass Spectrometer (Agilent). A ZORBAX Eclipse Plus C18 column (2.1 x 150mm, 1.8-Micron, Agilent) was used. The column temperature was maintained at 40 o C, and the solvent system was water containing 10mM ammonium acetate (pH 6.0, solvent A) and methanol ( Table S2. Synthesized cDNAs were then PCR amplified by Phusion TM High-Fidelity DNA Polymerase (New England Biolabs) with primers FW-GGAGGTGAGAGTGAGAGTAT and RV-TTCCCTTACCTACCACTTCC. The PCR products were first checked by 2 % agarose gel electrophoresis and then incubated with 4 units of Taq  I restriction enzyme (New England Biolabs) in 1 X CutSmart buffer (New England Biolabs) at 65 o C for 30 min. The restriction enzyme digestion products were also checked with 2 % agarose gel electrophoresis.

T-A cloning and Sanger sequencing
cDNA synthesized using methods above was PCR amplified by Phusion TM High-Fidelity DNA Polymerase with primers FW-GGAGGTGAGAGTGAGAGTAT and RV-TTCCCTTACCTACCACTTCC. The PCR products was purified by Zymo-Spin column and processed for Sanger sequencing.
For T-A cloning prior to sequencing, TOPOR TA Cloning R Kit (Thermo Fisher) was used. The PCR products from cDNA were cloned into TOPOR vector. We randomly picked 30 monoclones for each sample, and plasmids from these clones were prepared for Sanger sequencing with M13 primer.

NgTET1 oxidation, mTET1 oxidation and peroxotungstate reaction of m 5 C-RNA
NgTET1 was produced according to literature (Proc. Natl. Acad. Sci. U.S. A. 112, 4316-4321 (2015). For the oxidation of m 5 C-containing RNA2, 2 g RNA was incubated in 50 L solution containing 50 mM MOPS buffer (pH = 6.9), 100 mM ammonium iron (II) sulfate, 1 mM ketoglutarate, 2 mM ascorbic acid, 1 mM DTT, 50 mM NaCl, and 5 M NgTET1 at 37 o C for 1 hour. After that, 4 U of Proteinase K (New England Biolabs) was added to the reaction mixture and incubate at 37 o C for 30 min. The product was purified on Zymo-Spin column.
Mouse TET1 was purchased from . For the oxidation of m 5 C-containing RNA2, 2 g RNA was incubated in 50 L solution containing 50 mM HEPES buffer (pH = 8.0), 100 M ammonium iron (II) sulfate, 1 mM -ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, and 15 g mTET1 at 37 o C for 80 min. After that, 20 g of Proteinase K (New England Biolabs) was added to the reaction mixture and incubate at 50 o C for 1 hour. The product was purified on Zymo-Spin column.
15 L Oxidized m 5 C-containing RNA2 was then treated with 5 L 1 M aqueous sodium borohydride (Sigma) solution, in order to reduce the over-oxidized f 5 C to hm 5 C. After 1 hour of incubation at room temperature in the dark, the reaction was quenched by adding 10 L 750 mM sodium acetate (Sigma) (pH = 5.2). The product was purified on Zymo-spin column after no further gas was released. The peroxotungstate oxidation of the result RNA was performed according to the method described above. Then, cDNA was synthesized for this sample, and the PCR product was used for restriction enzyme digestion assay or analyzed by Sanger sequencing.

GT labelling of hm 5 C in RNA
Hm 5 C labelling was performed in 20 l solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl 2 , 200 μM UDP-Glc (New England Biolabs), and 10 U of βGT (Thermo Fisher), and 2 μg hm 5 C-containing RNA1 for 2 hours at 37 o C. The product was purified on Zymo-Spin column.

Cell culture and microRNA isolation
293T cells were maintained in high glucose DMEM medium (Gibco) at 37 o C under 5 % CO2 atmosphere. The media was supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin (Gibco).
Total microRNA which includes tRNA of the 293T cells was extracted from the cultured cells using PureLink TM miRNA Isolation Kit (Invitrogen). After measuring the concentration and checking the quality by agarose gel, the RNA was stored at -80 o C or proceed to next step.

NgTET1 oxidation, mTET1 oxidation and peroxotungstate reaction of tRNA from 293T cells
Typically, 2 g microRNA was used per reaction. To deacylate the tRNA, microRNA sample was first incubated in 50 L 0.5 M Tris-HCl, pH 9.0 at 37 o C for 1 hour. The product was purified on Zymo-Spin column. The NgTET1 oxidation, mTET1 oxidation and peroxotungstate reaction of deacylated tRNA were proceed using the method described above. Then, cDNA of the tRNA ASP(GUC) was synthesized using specific stem-loop primer, and the PCR product was analyzed by Sanger sequencing. Tables   Table S1. Oligonucleotides X = rC or m 5 C or hm 5 C.

Name
Type Sequence (5' to 3') Source   Figure S1. Hm 5 C conversion rate of different combinations of hm 5 C modified and unmodified RNA samples. Figure S2. Sanger sequencing results of PCR products from peroxotungstate treated hm 5 Ccontaining RNA2. Among 66 hm 5 C sites sequenced, 41 of them changed to T. The C-to-T conversion rate is 62.1 %. Figure S3. Sanger sequencing results of PCR products from peroxotungstate treated rC-containing RNA2 and m 5 C-containing RNA2. Among 33 sites sequenced in each sample, no C-to-T transition was detected, indicating the good selectivity of peroxotungstate oxidation on hm 5 C. Figure S4. Gel results of (1) control total RNA, (2) total RNA after bisulfite treatment and (3) total RNA after peroxotungstate treatment.