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. Overall design: Examination of gene expressive levels and m6A sites in control and mettl3 cKO mouse testes

采用Invitrogen公司的TRIzol试剂，依照制造商操作说明书，从对照组及Mettl3条件性敲除（conditional knockout, cKO）小鼠睾丸中分离总RNA。采用安捷伦生物分析仪2100（Agilent Bioanalyzer 2100）检测所有样本的RNA完整性，所有样本的RNA完整性数值（RNA Integrity Number, RIN）均高于9。
对于RNA测序（RNA-seq），取约2.5 μg总RNA，使用适配Illumina®平台的KAPA链特异性mRNA测序试剂盒（KAPABIOSYSTEMS，货号KR0960），依照制造商的实验流程完成文库制备。
对于m6A-miCLIP-seq，先取6 μg mRNA进行片段化，再以m6A抗体进行免疫沉淀，获取携带m6A修饰的RNA后，依照已发表的方法[Linder等人，2015；PubMed PMID: 26121403]构建cDNA文库。
所有文库均采用Illumina HiSeq3000平台以双端测序模式进行测序，生成101 bp长度的测序读段，并采用Illumina测序化学试剂v2完成测序反应。
实验整体设计：分析对照组与Mettl3条件性敲除小鼠睾丸中的基因表达水平及m6A修饰位点。