CK1-m6A
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), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified from 50g total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86 7min. The cleaved RNA fragments were incubated for 2h at 4 with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). 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 6003 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.