GSM6297302: cKO-2, IP; Mus musculus; RIP-Seq
Livers were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. For hepatocytes samples,primary hepatocytes were isolated as before(PMID:21633967), and RNA was harvested using Trizol reagent. MeRIP-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) according to the published procedure (Meyer et al., 2012) with slight modifications. Briefly, fragmented RNA was incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer for 2 hours at 4°C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 hours. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) by following the manufacturer's instruction. Purified RNA was used for RNA-seq library generation with NEBNext® Ultra™ RNA Library Prep Kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer.  For RNA-seq, the library was constructed as follows,   1, RNA isolation.Total RNA (1 μg) was used for removing the rRNAs using Ribo-Zero rRNA Removal Kits (Illumina, San Diego, CA, USA) following the manufacturer's instructions.2, Library Construction.RNA libraries were constructed by using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA,  USA) according to the manufacturer's instructions. Libraries were controlled for quality and quantified using  the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA).3, Sequencing.10 pM libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq Sequencer according to the manufacturer's instructions.