RNAseq analysis of the colon of intestine-specific adult Nkx2.2 mutant mice

Aim: Transcriptional analysis of the colon of adult Nkx2.2flox/flox;Villin-Cre (Nkx2.2int) mice versus control Methods: 2 cm of the colon (as measured after the caecum) of 6 week old control and mutant mice were dissected and total RNA extracted. Libraries were prepared from total RNA (RIN>8) with the TruSeq RNA prep kit (Illumina) and sequenced using the HiSeq2000 (Illumina) instrument. More than 20 million reads were mapped to the mouse genome (UCSC/mm9) using Tophat (version 2.0.4) with 4 mismatches and 10 maximum multiple hits. Significantly differentially expressed genes were calculated using DEseq. Results: 53 genes with a p-value <0.05 were down-regulated and 36 were up-regulated. Among the changed genes are enteroendocrine hormones. Conclusion: Nkx2.2 regulates enteroendocrine cell specification mRNA profiles of the colon of 6 week old control and Nkx2.2int mice were generated by deep sequencing, using Illumina HiSeq2000.研究目的：对成年Nkx2.2flox/flox;Villin-Cre（Nkx2.2int）小鼠的结肠组织与对照组开展转录组分析。 方法：选取6周龄对照组与突变体小鼠，截取盲肠后2 cm的结肠组织进行解剖分离，提取总RNA。采用TruSeq RNA建库试剂盒（Illumina公司）以RNA完整性指数（RIN）>8的总RNA构建测序文库，并使用HiSeq2000（Illumina公司）测序平台完成测序。利用Tophat（版本2.0.4）将超过2000万条测序读段（reads）比对至小鼠基因组UCSC/mm9版本，允许4个错配位点及最多10个多重比对位点。采用DEseq软件计算显著差异表达基因（differentially expressed genes）。 结果：共筛选得到53个下调表达基因（P值<0.05）及36个上调表达基因，其中包含肠内分泌激素相关基因。 结论：Nkx2.2可调控肠内分泌细胞的分化定型。本研究通过Illumina HiSeq2000平台开展深度测序，获取了6周龄对照组与Nkx2.2int小鼠的结肠组织mRNA表达谱。