GSM8486487: Male-Control1-ip; Mus; OTHER
Total RNAs were extracted with Trizol and NanoDrop 2000 spectrophotometer (ThermoFisher) was used to quantify the concentration of total RNA at a wavelength of 260 nm. About 1.5 µg of purified poly(A)-RNA was fragmented into ~100 nt fragments using RNA fragmentation buffer (AM8740, Thermo Scientific). One-tenth of fragmented RNAs were used for the construction of RNA-seq libraries (Input) and the left RNAs were used for the construction of m6A MeRIP-seq libraries (IP). m6A IP was conducted using an EpiMark® N6-Methyladenosine Enrichment Kit (#E1610S, NEB) following the product manual. The resultant m6A-enriched and untreated poly(A)-RNA fragments were used to construct strand-specific IP and Input libraries, respectively. A VAHTS® Universal V6 RNA-seq Library Prep Kit for Illumina (NR604-02, Vazyme Biotech. Co., Ltd.) and VAHTS® RNA Adapters set3 for Illumina (N809, Vazyme Biotech. Co., Ltd.) provided the necessary reagents for first- and second-strand cDNA synthesis, adaptor ligation, and library amplification. RNA-seq and MeRIP-seq of the prepared libraries were performed in Berry Genomics on the NovaSeq 6000 platform (Illumina, CA, USA) to obtain paired-end reads of 150 bp. Library quality was assessed on an Agilent Bioanalyzer 4200 TapeStation before loading onto the sequencer, ~8G of raw reads was obtained for each library.