GSM8354142: mESC_MeRIPseq.DMSO_rep1_IP; Mus musculus; OTHER
Total RNA was extracted and purified using Trizol reagent(Invitrogen) following the manufacturer's procedure. Poly(A) RNA was subsequently isolated from the total RNA using oligo dT beads (New England BioLabs, S1419S) to remove rRNA.The mRNA was then chemically fragmented into 150 nt fragments.Enrichment of m6A-modified mRNA was carried out using anti-m6A antibody (abcam, ab151230). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA).An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were 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. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol. MeRIP-seq