GSM8517663: Normal_P4_m6a_IP; Homo sapiens; OTHER
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RNA Integrity Number (RIN) >7.0. Poly(A) RNA is purified from 30μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, USA) using two rounds of purification. Then the poly(A)RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, USA) under 86℃ for 7min. Then the cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTP Solution (Thermo Fisher, USA). An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec, and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.