GSM3856428: Islet-2w.Input; Mus musculus; RIP-Seq
Islet total RNA was extracted using a RNeasy Micro kit (Qiagen) following the manufacturer's protocol.Briefly, 500 ug of fragmented mRNAs were incubated with 5 ug of anti- m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4) for 2 h at 4◦C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4◦C for an additional 2 h. Then, bound mRNAs were 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 mRNAs were used for RNA-seq library generation Purified mRNAs were used for RNA-seq library. Briefly, m6A RNA immunoprecipitation was performed with the GenSeq™ m6A-MeRIP Kit (GenSeq Inc., China) by following the manufacturer's instructions. Both the input sample without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads.with NEBNext® UltraTM RNA Library Prep Kit (NEB). Both the input sample (with- out immunoprecipitation) and the m6A IP sample were sub- jected to 150 bp paired-end sequencing on Illumina HiSeq sequencer. Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3′ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The reads were aligned to the reference genome (UCSC HG19) with Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified MACS software. Differentially methylated sites on RNAs were identified by diffReps. These peaks identified were mapped to transcriptome using home-made scripts.
GEO Accession
GSM3856428