RNA-seq from lung E18.5. 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. Overall design: mRNA profiles of E18.5 old wt and Mll3FDC/FDC (Mll3 KO) lungs were generated by deep sequencing in triplicates

本研究分别采集对照组与Mll3敲除（Mll3KO）小鼠各3份肺组织样本，通过mRNA表达谱分析评估了Mll3基因缺失对肺发育的影响。本次测序采用75碱基对（base-pair, bp）读长，每份样本获得3000万条测序读段，且各组唯一比对读段占比均维持在89%~91%的相近水平。所有生物学重复样本均按照基因型聚为两个清晰分离的类群，证实了本次测序数据质量可靠。为筛选差异表达基因，本研究针对该RNA-seq数据集应用了DESeq2分析工具。作为H3K4甲基转移酶，Mll3的预期调控特征与本次分析结果一致：在错误发现率（false discovery rate, FDR）≤5%的筛选标准下，下调表达的mRNA数量为上调表达的5倍。通过DAVID数据库开展基因本体（Gene Ontology, GO）功能富集分析结果显示，Mll3对细胞分化与形态发生的调控至关重要，进而参与肺脏成熟过程。借助基因集富集分析（Gene Set Enrichment Analysis, GSEA），本研究发现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。实验整体设计：对胚胎第18.5天（E18.5）的野生型（wild type, wt）及Mll3FDC/FDC（Mll3敲除）小鼠肺组织进行三次生物学重复的深度测序，以获取mRNA表达谱数据。