GSM6509747: NC-m6A-MeRIP-seq-IP3; Homo sapiens; RIP-Seq
For m6A-seq, total RNA was fragmented into 100-200 nt RNA fragments using ZnCl2 and incubated with anti-m6A polyclonal antibody (Sigma Aldrich) for 3 h at 4℃. Then mix the products above with protein A/G magnetic beads (Thermo Fisher) at 4 ℃ for an additional 2 h to obtain immunoprecipitated RNA fragments. The beads were then separated and washed with IP buffer three times. A NEBNext Ultra RNA library prep kit (NEB) was used to establish the library for the purified mRNA fragments. Each group was sequenced with an Illumina NovaSeq 6000 sequencer, and the single-end sequencing length was 150 bp. Reads were compared with human genome version GRCH38 through TopHat. If a gene had more than one subtype, the longest subtype was retained. ExomePeak (P < 0.01) was used to identify differential m6A-modified peaks between IP and input samples. RNA libraries were prepared for sequencing using standard Illumina protocols m6A-Seq