RNA-seq from lung E18.5

The impact of Mll3 removal on lung development was evaluated by mRNA profiling of three lung samples each from control and Mll3KO mice. Using 75-base-pair reads, 30 million reads per sample with comparable unique mapped reads (89-91%) were obtained. All biological replicates clustered into two well-separated groups according to genotype thereby lending confidence regarding data quality. To analyze differentially expressed genes, we applied DESeq2 analysis to the RNA-seq dataset. As expected for an H3K4 methyltransferase, we observed that 5 times more mRNAs were down- than up-regulated at a false discovery rate (FDR) of 5%. GO term analysis by DAVID revealed that Mll3 is important for the regulation of cell differentiation and morphogenesis, and therefore for the maturation of the lung. Using GSEA we concluded that the most affected cell type was the alveolar epithelial type-I cells involved in gas exchange. mRNA profiles of E18.5 old wt and Mll3FDC/FDC (Mll3 KO) lungs were generated by deep sequencing in triplicates本研究通过对对照组及Mll3敲除（Mll3KO）小鼠各3份肺组织样本进行mRNA表达谱分析，评估了Mll3缺失对肺发育的影响。测序采用75碱基对（75-base-pair）读长，每份样本获取约3000万条测序读段，且各组样本的唯一比对读段占比均稳定在89%~91%区间。所有生物学重复样本均按照基因型聚为两个界限清晰的组别，证实了本次测序数据的质量可靠性。为分析差异表达基因，本研究针对该RNA测序（RNA-seq）数据集使用DESeq2工具进行差异分析。作为H3K4甲基转移酶（H3K4 methyltransferase）的典型特征，本研究观察到在错误发现率（false discovery rate, FDR）为5%的筛选阈值下，下调表达的mRNA数量是上调表达的5倍。通过DAVID数据库开展基因本体（Gene Ontology, GO）术语富集分析，结果显示Mll3在细胞分化与形态发生的调控中发挥关键作用，进而参与肺脏成熟过程。通过基因集富集分析（Gene Set Enrichment Analysis, GSEA），本研究确定受影响最显著的细胞类型为参与气体交换的I型肺泡上皮细胞。本研究还通过三重重复深度测序，获取了胚胎发育第18.5天（E18.5）野生型（wt）及Mll3FDC/FDC（Mll3敲除）小鼠肺组织的mRNA表达谱。