Systemic determination of PRDM16 regulatory genes in the aorta

We determined the differentially expressed genes in the thoracic aorta (TA) from vascular smooth muscle cell (VSMC)-specific Prdm16 knockout (Prdm16SMKO) and control mice by RNA-sequencing (RNA-seq) analysis. We also performed chromatin immunoprecipitation (ChIP)-sequencing (ChIP-seq) analysis to identify potential target genes of PRDM16. By overlapping the RNA-seq and ChIP-seq dataset, we are able to identify the target genes of PRDM16 in the aorta and investigate the roles of PRDM16 in the vascular biology. Overall design: The thoracic aorta from Prdm16SMKO and control mice were harvested and subjected to RNA-seq analysis. The differentially expressed genes between Prdm16SMKO and control mice were analyzed. For ChIP-seq analysis, mouse fibroblast cells were infected with lentivirus carrying flag-tagged PRDM16 or vector (as control) for 48 hours and were selected with 2 ug/mL puromycin for 1 week, followed by ChIP using anti-flag antibody. The purified DNA was subjected to ChIP-seq.

本研究通过RNA测序（RNA-sequencing, RNA-seq）分析，鉴定了血管平滑肌细胞（vascular smooth muscle cell, VSMC）特异性Prdm16敲除（Prdm16SMKO）小鼠与对照小鼠胸主动脉（thoracic aorta, TA）中的差异表达基因。同时通过染色质免疫共沉淀测序（chromatin immunoprecipitation-sequencing, ChIP-seq）分析，筛选PRDM16的潜在靶基因。通过将RNA-seq与ChIP-seq数据集进行交集分析，本研究得以鉴定主动脉中PRDM16的靶基因，并探究PRDM16在血管生物学中的作用。
总体实验设计：收集Prdm16SMKO小鼠与对照小鼠的胸主动脉，进行RNA-seq分析，并对两组小鼠的差异表达基因进行分析。针对ChIP-seq实验，将携带flag标签PRDM16的慢病毒或空载对照病毒感染小鼠成纤维细胞，感染48小时后采用2 μg/mL嘌呤霉素筛选1周，随后使用抗flag抗体进行染色质免疫共沉淀，将纯化得到的DNA进行ChIP-seq测序。