GSM5234413: Input_PCIF1 KO_rep2; Homo sapiens; OTHER
Total RNA was extracted using TRIzol. About 100mg RNA were then used to isolate poly(A) RNA using two rounds of oligo25 Dynabeads capture. For one sample, 100 μg poly(A) RNA was fragmented in fragmenting buffer at 70°C for 15min. Fragmentation is stopped by the addition of EDTA to a final concentration of 70 mM. mRNA is purified  with the RNA Clean and Concentrator 5 kit (Zymo Research). Uncapped and fragmented transcripts are phosphorylated by treating the mRNA with 200 U of T4 PNK (NEB) in T4 ligase buffer for 1:30 h at  37°C. Then, 20 U of Terminator 5'-Phosphate-Dependent Exonuclease (Lucigen) is added with 1X Exonuclease buffer A. Samples are treated for 3 h at 30°C to remove phosphorylated (ie. uncapped and sheared) transcripts. The RNA enriched for 5'-capped transcripts is purified with the RNA clean and concentrator 5 kit (Zymo Research). 30 U of Cap-Clip (CellScript) are added and incubated for 2 h at 37°C in CAP-CLIP buffer to remove the cap. RNA is again purified with the RNA clean and Concentrator 5 kit. 10% of the treated RNA is saved as input, the rest is diluted and incubated with 5 μg of anti-m6A antibody (abcam, ab151230) at 4 °C for 2 h. 100 μl protein G magnetic beads were added to pull down the m6A immunoprecipitation complex. After wash for three times, the bound RNA was eluted in 100 μl elution buffer and purified using RNA clean and concentrator-5 kit. Final libraries amplified and were subjected to paired-end sequencing in the HT sequencing IGM Genomics core, UCSD.