GSM9008975: WT1_IP; Mus musculus; OTHER
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel.Approximately more than 1μg of total RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) at 94℃ for 5 min.Then the cleaved RNA fragments（including rRNA fragments） were incubated with m6A antibody - dynabead compounds. Then the IP RNA was reverse-transcribed to create the first-strand cDNA by SMARTScribe™ Reverse Transcriptase (CloneTech，cat.634414，Japan)which were next used to add adapter and synthesise second-stranded DNAs with PCR by the following conditions:initial denaturation at 94℃ for 1 min; denaturation at 98℃ for 15 sec, annealing at 55℃ for 15 sec, and extension at 68℃ for 30 sec; and then final extension at 68℃ for 2 min,5 cycles. Then the amplified DNA-seq library is purified by immobilization onto pure beads. The cDNA sequences originating from rRNA reverse transcription were cut by ZapR v2 and R-Probes v2（for mammal） under the condition :incubation at 72℃ for 2 min,4℃ for 2 min，37℃ for 1h,72℃ for 10 min，4℃ forever. The final library was amplified by second round PCR which consistent with the first round PCR program,and the number of PCR cycles increased to 12-16. At last, we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.