RNA-seq analysis of testis transcripts from Wt and Trf2-/- mice [RNA-seq]

TRF2 is a paralogue of TATA-box binding protein (TBP) with highest expression in testis. Although Trf2 inactivation in mice leads to arrested spermatogenesis, there is no direct evidence that Trf2 is recruited to chromatin to directly regulate gene expression. We used genetically modified mice where endogenous Trf2 has been modified to carry a TAP-TAG to perform ChIP-reChIP followed by deep sequencing. We found that Trf2 is recruited to all active promoters as a subunit of TFIIA/ALF complex together with TBP. To assess the effect of Trf2 inactivation on gene expression we performed RNA-seq on WT and Trf2-/- testes at 21 days of age when haploid cell gene expression is activated. The testes from three 21 day old WT and three Trf2-/- males were taken to prepare total RNAs for deep sequencing.TRF2是TATA盒结合蛋白（TATA-box binding protein, TBP）的旁系同源物，在睾丸中表达水平最高。尽管小鼠体内Trf2基因失活会导致精子发生停滞，但目前尚无直接证据表明Trf2被招募至染色质以直接调控基因表达。本研究采用内源Trf2被改造为携带TAP-TAG的基因工程小鼠，开展染色质免疫沉淀-再染色质免疫沉淀（ChIP-reChIP）实验并结合深度测序。结果发现，Trf2作为TFIIA/ALF复合物的亚基，与TBP共同被招募至所有活跃的启动子区域。为评估Trf2失活对基因表达的影响，本研究在单倍体细胞基因表达被激活的21日龄阶段，对野生型（wild type, WT）和Trf2敲除（Trf2-/-）的睾丸组织进行RNA测序（RNA-seq）。研究采集了3只21日龄野生型雄鼠与3只Trf2敲除雄鼠的睾丸组织，提取总RNA用于深度测序。