RNA-sequencing of mouse intestinal stem cells (ISCs)

We previously demonstrated that SRCAP regulates the self-renewal of ESCs and modulates lymphoid lineage commitment. We further validated that SRCAP was mainly distributed in liver, spleen and intestine by Northern blot. SRCAP was also highly expressed in Lgr5+ ISCs. We then sought to explore the physiological role of SRCAP in the regulation of self-renewal maintenance of ISCs.We then generated Srcapflox/flox mice through insertion of loxP sequences flanking at the exon5 of Srcap gene locus. We established Srcapflox/flox;Lgr5GFP-CreERT2 mice through crossing Srcapflox/flox mice with Lgr5GFP-CreERT2 mice. With administration of tamoxifen (TAM), Srcap was completely deleted in Lgr5+ ISCs. We identified that Srcap deficiency impairs the self-renewal of ISCs and intestinal epithelial regeneration. Through RNA-sequencing, we sough to identify the key gene regulated by SRCAP in ISC self-renewal. Overall design: Total RNAs were isolated from WT or SRCAP-deficient ISCs and used for RNA sequencing

本团队此前已证实，SRCAP可调控胚胎干细胞（Embryonic Stem Cells, ESCs）的自我更新，并调控淋巴谱系定向分化。本研究进一步通过Northern印迹（Northern blot）实验验证，SRCAP主要分布于肝脏、脾脏与肠道组织中；同时，SRCAP在Lgr5阳性肠干细胞（Intestinal Stem Cells, ISCs）中亦呈高表达状态。据此，本研究旨在探究SRCAP在肠干细胞自我更新维持过程中的生理学功能。本研究通过在Srcap基因座的外显子5两侧插入loxP序列，构建了Srcap条件性敲除（flox/flox）小鼠；将该小鼠与Lgr5GFP-CreERT2小鼠杂交，获得Srcap flox/flox;Lgr5GFP-CreERT2双阳性小鼠。经他莫昔芬（Tamoxifen, TAM）给药处理后，可在Lgr5阳性肠干细胞中完全敲除Srcap基因。研究发现，Srcap基因缺失会损伤肠干细胞的自我更新能力，并抑制肠道上皮再生过程。本研究通过RNA测序（RNA-sequencing）技术，旨在筛选鉴定SRCAP在肠干细胞自我更新过程中调控的关键靶基因。实验整体设计：从野生型（Wild Type, WT）或SRCAP基因缺失型肠干细胞中提取总RNA，用于RNA测序实验。