AC16-ALKBH5-OE
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 25 g of total RNA representing a specific adipose type was used to deplete ribosomal RNA according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA). Following purification, the ribosomal-depleted RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86 7min. Then 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). Then the IP RNA was reverse-transcribed to create the cDNA by SuperScript II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were 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 is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are 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 are 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. The average insert size for the final cDNA library was 30050 bp. At last, we performed the 2150bp paired-end sequencing (PE150) on an illumina Novaseq 6008 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol