title: GSM9008979: CK1_IP; Mus musculus; OTHER
library_construction_protocol: 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.