GSM7765082: H9 cells, Ctrl_m6A_IP; Homo sapiens; OTHER
Total RNAs were extracted from METTL3-SMASh tagged hNPCs (treated with or without ASV) using TRIzol Reagent (Invitrogen, 15596026). DNA digestion was car-ried out after RNA extraction by DNaseI. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally quan-tified by Qubit3.0 with QubitTM RNA Broad Range Assay kit (Life Technologies, Q10210). 50 μg total RNAs were used for polyadenylated RNA enrichment by VAHTS mRNA Capture Beads（VAHTS, N401-01/02）. 20mM ZnCl2 was added to mRNA and incu-bated at 95℃ for 5min-10min until the RNA fragments were mainly distributed in 100-200 nt. Then 10% RNA fragments were saved as “Input” and the rest was used for m6A immunoprecipitation（IP）.The specific anti-m6A antibody (Synaptic Systems, 202203, 2.5 μg / IP) was applied for m6A immunoprecipitation. RNA samples of both input and IP were prepared using TRIzol reagent (Invitrogen, 15596026). The stranded RNA sequencing library was constructed by KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Wuhan Seqhealth, DR08502) following the manufacturer's in-struction. The kit eliminated duplication bias in PCR and sequencing steps, by using unique mo-lecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules. The library products corresponding to 200-500 bps were enriched, quantified and fi-nally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model. MeRIP-seq