GSM4677601: Heart_NC_2 weeks_IP; Mus musculus; OTHER
Total RNAs were extracted from CHAPIR mimic and negative control (NC) mice hearts using Trizol reagent (Invitrogen). RNA were quantified using a NanoDrop ND-1000 instrument. mRNAs were isolated from total RNA sample and they were chemically fragmented to 100-nucleotides-long fragments. MeRIP was performed to enrich m6A methylated mRNAs using Anti-N6-methyladenosine (m6A) antibody (Abcam, ab208577). We used KAPA Stranded mRNA-seq Kit (Illumina) for RNA-seq library preparation of both m6A enriched RNAs and input mRNAs. The libraries were subjected to denaturation to obtain single-stranded DNA molecules and captured on Illumina flow cells. Then, they were amplified in situ as sequencing clusters and sequenced for 150 cycles on Illumina HiSeq 4000 system as per the manufacturer's instructions. The image analysis and base calling were carried out using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). The sequencing quality was examined by FastQC software and trimmed reads (pass Illumina quality filter, trimmed adaptor bases by cutadapt) were aligned to genome sequences from Ensembl using HISAT2 software (v2.1.0). The aligned reads were used for peak calling by exomePeak and a statistically significant MeRIP enriched regions (peaks) were identified for each transcript and compared by exomePeak. The MeRIP enriched regions (peaks) were annotated by the overlapped gene using the newest version of Ensembl database. Then the statistical analysis of m6A peak in each transcript region was done.
GEO Accession
GSM4677601