GSM7816676: tNCC_m6A_rep1; Homo sapiens; RIP-Seq
RNA for RNA-seq and m6A RIP-seq was extracted from cells using using TRIZOL reagent (ThermoFisher Scientific, 15596026) and Direct-zol RNA Miniprep (ZYMO research, R2050). For ChIP-seq cells were fixed with formaldehyde (1% final concentration) for 15 min at RT. Fixed cells were washed with cold PBS, lysed and the chromatin was sheared using a Bioruptor (Diagenode) until an average fragment size range of 200-500 bp was achieved. Immunoprecipitation of solubilized chromatin was carried out with 3 µg of METTL3 MYCN / H3K27ac antibody overnight at 4°C. The immunoprecipitated complex was captured using a mix of Protein A and G Dynabeads (Invitrogen), washed and RNase A treated before eluting the DNA. The ChIP DNA was directly used to make libraries. m6A-RIP-seq: RNA was isolated using TRIZOL reagent (Invitrogen). RNA fragmentation followed by m6A-RIP was performed with 15ug of RNA spiked-in with 9 ng of Bacterial RNA. Sequencing libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v2 (Pico Input Mammalian) from Takara and single-end sequenced (1X 88 bp) on Illumina NextSeq 2000 platform. RNA-seq: Short-insert libraries were prepared at BGI Tech Solutions (Hong Kong) for strand specific mRNA sequencing on a DNBSEQ platform with a PE100 sequencing read length. ChIP-seq: ChIP-seq libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, E7645L following manfactures protocol.