title: m6A-seq Yy1 cKO-IP-1
design: 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 1g of total RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) at 94 for 5 min. The cleaved RNA fragments (including rRNA fragments) were then incubated with m6A antibody-dynabead compounds. The Input RNA and IP RNA were reverse-transcribed to create the first-strand cDNA by SMARTScribe Reverse Transcriptase (CloneTech, cat.634414, Japan), which were used to perform m6A-qPCR. Primers used in this study are listed in Table S8. The above first-strand cDNA was attached with adapter and synthesized 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. The amplified DNA-seq library was 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, finally stored at 4C. The final library was amplified by second round PCR which was consistent with the first round PCR program (PCR cycles: 13). Finally, the 2150bp paired-end sequencing (PE150) was performed on an illumina Novaseq 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.