Transcriptome analysis of gene expression and single-nucleotide-resolution m6A sites in normal and mettl3 cKO testis samples (CLIP-Seq)

RNA was isolated from control and Mettl3 cKO mouse testes using the TRIzol (Invitrogen) reagent by following the manufactures's instructions. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. For RNA-seq, approximately 2.5 µg of total RNA was then used for library preparation using a KAPA Stranded mRNA-Seq Kit Illumina® platform (KAPABIOSYSTEMS, KR0960) according to the manufacturer’s protocol.For m6A-miCLIP-seq, 6 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403].All the libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used. Examination of gene expressive levels and m6A sites in control and mettl3 cKO mouse testes