GSM5293192: m5C_WT_Total-RNA_VSV-infected_BS-treated_2; Homo sapiens; OTHER
For virus-infected or uninfected A549 wild-type and NSUN2 knockout cells, cellular total RNA was isolated with TRIzol reagent (Invitrogen) following manufacturer's protocol by isopropanol precipitation.   When extracting rRNA-depleted total RNA for sequencing m5C in RNA, RiboMinus™ Eukaryote System v2 (Invitrogen™) was further used for rRNA removal. When extracting polyA+ RNA for sequencing m5C in RNA, Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™) was used for polyA+ RNA enrichment. RSV-infected or VSV-infected NSUN2 knockout and wild-type A549 cells were prepared as described (2 replicates each, one 10-cm plate per replicate). After extracting the rRNA-depleted total RNA, ~200 ng RNA was fragmented into 50-70 nt length using Na2CO3/NaHCO3 buffer (pH 9.2) and heated at 95 °C for 3 min, followed by purification with Oligo Clean & Concentrator (Zymo Research). ~50 ng was saved as “input” and the remainder was subjected to EZ RNA Methylation Kit (Zymo Research) for bisulfite treatment. 3'- and 5'-end repair was conducted for “input” and bisulfite-treated RNA by T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific). RNA was mixed with 5 µL of 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 µL of T4 PNK, diluted to a final volume of 50 µL, and incubated at 37 °C for 1 hour; then 2 µL of T4 PNK and 5 µL 10 mM ATP were added for another incubation 37 °C for 30 min, followed by Oligo Clean & Concentrator purification. To perform 3'-adaptor ligation, ~6 µL repaired RNA fragments were incubated with 1.0 µL 3'-Adaptor (5'App-NNNNN ATCACGAGATCGGAAGAGCACACGTCT-3SpC3) at 70 °C for 2 mins and immediately placed on ice. 10 µL 3´ Ligation Reaction Buffer (2X) (NEB) and 3 µL 3´ Ligation Enzyme Mix (NEB) were added to the RNA-adapter mixture. The reaction was incubated at 25 °C for 2 hours followed by 16 °C for 10 hrs. Then 1.0 µL SR RT Primer for Illumina (NEB) was added to the mixture, followed by 5 minutes at 75°C, 15 minutes at 37°C, 15 minutes at 25°C, and then on ice. The denatured 1.0 µL 5'-Adaptor (5'-GUUCAGAGUUCUACAGUCCGACGAUCNNNNN-3') was added into the mixture, together with 1.0 µL 5´ Ligation Reaction Buffer (10X) (NEB) and 2.5 µL 5´ Ligation Enzyme Mix (NEB). The reaction was incubated at 25 °C for 4 hours, followed by RNA Clean & Concentrator purification  The purified RNA was mixed with 4 µL 5X SSIV Buffer (Thermo Scientific), 2 µL of 10 mM dNTP Solution Mix (NEB), 1 µL 100 mM DTT, 1 µL of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Scientific), and 1 µL of SuperScript IV Reverse Transcriptase (Thermo Scientific). The reaction was diluted to 20 uL and incubated at 50 °C for 30 mins. 1.0 µL of RNase H (NEB, M0297L) was then added and the reaction was incubated at 37 °C for 20 mins. The cDNAs were purified by Oligo Clean & Concentrator (Zymo Research). The libraries were amplified using the SR Primer for Illumina (NEB) and indexed primers (from NEBNext Multiplex Oligos for Illumina). RNA-Seq. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.