RNA-sequencing from wild-type (WT) or Tie2-Ino80 knockout (KO) mouse embryo hearts

Purpose: Endothelial cell-specific knockout of the INO80 chromatin-remodeling complex in developing mouse embryos results in defective coronary angiogenesis. Transcriptome analysis on whole hearts was performed to understand how Ino80 regulates the genome to influence angiogenesis. Methods: mRNA was extracted from whole hearts after surgical removal from embryonic day 13.5 mice, either WT or Tie2-Ino80 KO, and prepped for Illumina sequencing using the NEBNext Ultra RNA Library Prep kit. Results: Deletion of Ino80 in the two major coronary progenetiors results in intermediate non-compaction phenotypes and an increase in E2F-mediated gene expression and cellular proliferation. Conclusions: Ino80 normally functions to suppress E2F-mediated proliferation in cardiac endothelial cells in order to promote productive angiogenesis and prevent underdevelopment of the myocardium heart muscle. Loss of this critical chromatin-remodeling function results in human disease phenotypes. mRNA profiles from embryonic day E13.5 mice were generated from WT or Tie2-Ino80-KO whole hearts using deep sequencing with three biological replicates for each. An average of 33.9M and 53.5M reads per replicate were included in the final analysis for the WT and KO samples, respectively.研究目的：在发育中的小鼠胚胎内特异性敲除内皮细胞的INO80染色质重塑复合物（INO80 chromatin-remodeling complex），会引发冠状动脉血管生成缺陷。本研究对完整心脏开展转录组分析，以阐明Ino80如何通过调控基因组来影响血管生成过程。 研究方法：从胚胎发育第13.5天（E13.5）的野生型（Wild Type, WT）或Tie2特异性Ino80敲除（Tie2-Ino80 KO）小鼠体内手术摘取完整心脏，提取总信使RNA（mRNA），使用NEBNext Ultra RNA文库制备试剂盒构建测序文库，随后进行Illumina测序。 研究结果：在两种主要冠状动脉祖细胞中敲除Ino80，会导致中度非致密化表型，并上调E2F介导的基因表达与细胞增殖水平。 研究结论：Ino80在心脏内皮细胞中正常发挥抑制E2F介导的细胞增殖的功能，从而促进有效的冠状动脉血管生成，防止心肌（myocardium）发育不全。这种关键染色质重塑功能的缺失会引发人类疾病表型。本研究通过深度测序，对E13.5天胚胎小鼠的野生型或Tie2-Ino80-KO完整心脏进行mRNA表达谱分析，每组设置3次生物学重复。野生型样本与敲除样本的每个生物学重复平均测序读段数分别为33.9M与53.5M，最终均纳入统计分析。