title: GSM8995384: MOLM13, nascent RNA, 16hr 5azaC, rep2; Homo sapiens; RNA-Seq
library_name: GSM8995384
library_construction_protocol: For RNA-seq, total RNA was harvested by TRIzol™ reagent. For nascent RNA-seq, EU labeled RNA was enriched following manufacturer's instructions with Click-iT™ Nascent RNA Capture Kit for gene expression analysis (Invitrogen™, C10365). After the final washing steps, RNA was eluted in 50 μL of 95% formamide supplemented with 2.5 mM EDTA by heating at 65°C for 5 mins, followed by 95°C for 5 mins. The supernatant was immediately harvested while still hot, and RNA was extracted using 1 mL TRIzol™ reagent. For m5C MeRIP, chromatin-associated RNA was purified. For MOLM13 cells, 0.02 million drosophila S2 cells were added to 20 million MOLM13 cells before proceeding with cellular fractionation. rRNA removal was carried out using RiboMinus Eukaryote System v2 (Invitrogen™, A15026). 2 μg ribo-depleted caRNA was then used for m5C immunoprecipitation. RNA was fragmented using 1× fragmentation buffer (NEB, E6186A) at 94°C for 2 mins, and 5% of the sample was saved as input. The RNA was heated to 72°C for 2 mins and then placed immediately on ice for denaturation. Next, the RNA was added to protein G beads conjugated with 2 μg anti-m5C antibody (Diagenode, MAb-081-100) in 300 μL IP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Triton X-100, 1 mM spermidine, 0.2 U/μL SUPERase•In™ RNase Inhibitor), and incubated with end-to-end rotation at 4°C for 2 hours. The beads were washed three times with IP buffer and eluted with buffer RLT (Qiagen, 79216). For CLIP-seq, 100 million cells were used for each replicate. Following the designated treatment, cells were crosslinked in PBS using 4500 J/m2 UV at 254 nm. Cells were lysed in 200 μL iCLIP lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40 substitute, 0.1% SDS, 0.5% sodium deoxycholate, 100× proteinase inhibitor cocktail and 0.2 U/μL SUPERase•In™ RNase Inhibitor) at 4°C for 15 mins with end-to-end rotation. SDS were then added to reach a final concentration of 1% for protein release from chromatin. Samples were sonicated (35% amplitude, 2s on / 4s off cycles, 1 min on ice) and diluted 10-fold with SDS-free iCLIP lysis buffer to reduce SDS back to 0.1%. After centrifugation at 15,000 x g for 30 mins at 4°C, the supernatant was harvested and treated with 0.2 U/μL RNase T1 (Thermo Scientific™, EN0642) at 22°C for 15 mins, then quenched on ice for 5 mins. 2% of the lysate was saved as input and digested with 10 U/μL RNase T1 at 22°C for 10 mins, followed by proteinase K digestion, RNA purification and end repair. The remaining lysate was incubated overnight at 4°C with protein A beads conjugated to 30 μL MBD6 antibodies (gifted from Prof. Lu Wang) per sample. Beads were washed three times with CLIP wash buffer (50 mM Tris-HCl pH 7.5, 300 mM KCl, 0.05% NP-40, 1000× protein inhibitor cocktail and 0.2 U/μL SUPERase•In™ RNase Inhibitor), followed by digestion with 10 U/μL RNase T1 at 22°C for 10 mins. Beads were further washed for three times each with CLIP high salt buffer (50 mM Tris-HCl pH 7.5, 500 mM KCl, 0.05% NP-40, 100× protein inhibitor cocktail and 0.2 U/μL SUPERase•In™ RNase Inhibitor) and PNK buffer without DTT, and subjected to end-repair using T4 PNK (NEB, M0201L). After proteinase K digestion, RNA was purified for library preparation. The purified RNA was constructed for library with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian, with ribominus reactions supplied by the kit. For the m5C MeRIP-seq, customized adapters with umi was used for deduplication purpose. For CLIP-seq, libraries were constructed using the NEBNext® Small RNA Library Prep Set.