RNAseq for CDK7 brown adipose tissue specific knockout mice under room temperature and 6 hrs cold exposure.

Purpose: The goal of this study are to compare global gene expression profile in brown adipose tissue in Cdk7 bKO and WT control mice at both RT and after 6 hours cold exposure Methods: Interscapular BAT pads were taken from 12-week-old CDK7 bKO and control littermates either at room temperature or after 6 hours of cold exposure with food. Mice were sacrificed at zeitgeber time 9. Total RNA was extracted with TRI Reagent (Sigma). Two RNA samples from two mice with same genotype and condition were pool together (each 250 ng) to constitute 500 ng of total RNA from biological triplicates (three WT-RT, three CDK7 bKO-RT, three WT-cold, and three CDK7 bKO-cold) for sequencing. Results: Reads were also aligned to the Mus musculus.GRCm38.86 transcriptome using STAR (v. 2.5.2b, Dobin et al. 2013) and the estimation of the isoforms abundance was computed using RSEM (v. 1.2.31, Li and Dewey 2011). Conclusions: Differentially expressed genes (DEGs) were identified between genotype and treatment. Interscapular BAT pads were taken from 12-week-old CDK7 bKO and control littermates either at room temperature or after 6 hours of cold exposure with food. Mice were sacrificed at zeitgeber time 9. Total RNA was extracted with TRI Reagent (Sigma). Two RNA samples from two mice with same genotype and condition were pool together (each 250 ng) to constitute 500 ng of total RNA from biological triplicates (three WT-RT, three CDK7 bKO-RT, three WT-cold, and three CDK7 bKO-cold) for sequencing.研究目的：本研究旨在比较室温（room temperature, RT）及6小时冷暴露条件下，Cdk7基因敲除（Cdk7 bKO）小鼠与野生型（wild type, WT）对照小鼠棕色脂肪组织（brown adipose tissue, BAT）的全基因表达谱。 实验方法：从12周龄的CDK7 bKO小鼠及其同窝野生型对照小鼠肩胛间采集棕色脂肪组织（BAT）垫，分别在室温条件下，或正常进食状态下经6小时冷暴露后取材。所有小鼠均在授时因子时间（zeitgeber time, ZT）9时处死。采用Sigma公司的TRI试剂（TRI Reagent）提取总RNA。将同基因型、同处理条件的两只小鼠的RNA样本各250 ng混合，得到500 ng总RNA，共设置4组生物学重复样本各3份（WT-RT组、CDK7 bKO-RT组、WT-冷暴露组、CDK7 bKO-冷暴露组各3份）用于测序。 测序分析：使用STAR（v.2.5.2b, Dobin et al. 2013）将测序读段比对至小家鼠（Mus musculus）GRCm38.86转录组，并通过RSEM（v.1.2.31, Li and Dewey 2011）计算转录本丰度。 研究结论：本研究鉴定了不同基因型与处理条件下的差异表达基因（differentially expressed genes, DEGs）。实验取材流程如下：从12周龄CDK7 bKO小鼠及同窝野生型对照小鼠肩胛间采集棕色脂肪组织（BAT）垫，分别在室温或正常进食下6小时冷暴露后取材；所有小鼠均于授时因子时间9处死，采用Sigma公司的TRI试剂（TRI Reagent）提取总RNA；将同基因型、同处理条件的两只小鼠的RNA样本各250 ng混合为500 ng总RNA，共设置4组生物学重复样本各3份（WT-RT、CDK7 bKO-RT、WT-冷暴露、CDK7 bKO-冷暴露各3份）用于测序。