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

RNA was isolated from control and Mettl3 cKO mouse testes using the TRIzol (Invitrogen) reagent by following the manufacturer'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采用Invitrogen公司的TRIzol试剂，按照制造商说明书从对照组及Mettl3条件性敲除（Mettl3 cKO）小鼠睾丸中分离总RNA。所有样本均采用Agilent Bioanalyzer 2100检测RNA完整性，所有样本的RNA完整性数值（RIN，RNA integrity number）均高于9。针对RNA测序（RNA-seq，RNA sequencing），取约2.5 μg总RNA，按照制造商方案，使用KAPA Stranded mRNA-Seq Kit Illumina®平台（KAPABIOSYSTEMS，KR0960）进行文库构建。针对m6A-miCLIP-seq实验，先取6 μg mRNA进行片段化，再使用m6A抗体进行免疫沉淀，将获得的含m6A的RNA按照已报道的方法[Linder等人，2015；PMID: 26121403]进行cDNA文库构建。所有文库均采用Illumina HiSeq3000平台进行双端测序，读取长度为101 bp，使用Illumina测序化学试剂v2。对对照组与Mettl3 cKO小鼠睾丸的基因表达水平及m6A修饰位点进行了检测分析。