Gene expression profile in Ronin (Thap11) conditional knock out retina through RNA-sequencing

Conditional knockout of the transcription factor Ronin (Thap11) in retinal progenitor cells (RPCs) results in a profound failure cell proliferation resulting in a hypoplastic adult retina that also suffers from photoreceptor degeneration. The goal of this study was to determine which genes are deregulated in response to loss of Ronin transcription factor activity in the developing retina. We generated Ronin flox/flox (Control) and Chx10-Cre::GFP+/tg; Ronin flox/flox (CKO) mice, in which Ronin loss occurs specifically within RPCs, and performed RNA-Seq analysis of embryonic day E14.5 (E14.5) retinae. Three independent pools of control and Ronin CKO retinae were collected consisting of a minimum of 10 retinae per pool and total RNA was extracted followed by polyA selection, fractionation (200-500 nucleotide range) and generation of cDNA. The resulting DNA was then used for standard Illumina adaptor ligation and sequencing. This experiment revealed decreased expression of a large group of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Retinal mRNA profiles of embryonic day 14.5 (E14.5) Control (Ronin flox/flox) and Ronin CKO (Chx10-Cre::GFP+/tg; Ronin flox/flox) mice were generated by deep sequencing, in triplicate using Illumina HiSeq2500在视网膜祖细胞（retinal progenitor cells, RPCs）中对转录因子Ronin（Thap11）进行条件性敲除，会导致严重的细胞增殖障碍，进而形成发育不全的成年视网膜，同时还会引发光感受器变性。本研究旨在明确发育中的视网膜内转录因子Ronin活性缺失后，哪些基因的表达发生了失调。我们构建了Ronin flox/flox（对照组）以及Chx10-Cre::GFP+/tg; Ronin flox/flox（条件性敲除组，CKO）小鼠，该CKO模型中Ronin仅在RPCs中发生缺失，并对胚胎第14.5天（E14.5）的视网膜进行了RNA测序（RNA-Seq）分析。我们收集了3组独立的对照及Ronin CKO视网膜样本，每组至少包含10个视网膜；提取总RNA后，通过polyA富集、片段筛选（区间为200-500核苷酸）并合成cDNA。所得DNA用于标准的Illumina接头连接及测序实验。本实验发现，大量线粒体基因（包括电子传递链（electron transport chain, ETC）的组分）的表达出现下调，而这些基因近期被证实为细胞周期的直接调控因子。本研究采用Illumina HiSeq2500平台，对3次生物学重复的胚胎第14.5天（E14.5）对照（Ronin flox/flox）及Ronin CKO（Chx10-Cre::GFP+/tg; Ronin flox/flox）小鼠视网膜进行深度测序，获得其mRNA表达谱。