Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type Mice and Toll-like Receptor 4 Intestinal Epithelial Conditional Knockout Mice Ileal Transcriptomes

Toll-like receptor 4 (TLR4), the receptor of gram-negative bacterial endotoxin, is present on the intestinal epithelium and plays pivotal roles in intestinal epithelial differentiation and host defense. To better understand TLR4 on the intestinal epithelium, the ileum samples from wild type mice and mice lacking TLR4 in the intestinal epithelium (TLR4deltaIEC) were subjected to RNA-seq analysis to interrogate the transcriptome. We mapped about 20 million sequence reads per sample to the mouse genome and identified 86477 genes in the ileum of wild type and TLR4deltaIEC mice. 455 genes were differentially expressed between wild type and TLR4deltaIECmice and 1037 genes were absent either from wild type or TLR4deltaIEC mice with G-factor value 0.015 and delta cutoff value 0.54. Pathway analysis via IPA indicated some impacted canonical KEGG pathways such as DNA replication, PPAR signaling, Toll-like receptor signaling, cytokine-cytokine interaction, adherens junction, Jak-STAT signaling, and tight junction. Total RNA was isolated from the ileum of 3 wild type mice and 3 TLR4deltaIEC mice, and a barcode was added to the sample from each individual mouse during the whole transcriptome library preparation. The pooled samples from 3 wild type mice (barcodes BC01, BC02 and BC03) and from 3 TLR4deltaIEC mice (barcodes BC07, BC08 and BC09) were subjected to RNA sequencing, respectively.Toll样受体4（Toll-like receptor 4, TLR4）是革兰氏阴性细菌内毒素的受体，表达于肠上皮细胞，在肠上皮分化与宿主防御过程中发挥关键作用。为深入探究肠上皮TLR4的生物学功能，研究人员对野生型小鼠及肠上皮特异性TLR4敲除小鼠（TLR4ΔIEC）的回肠样本开展RNA测序（RNA-seq）分析以解析其转录组特征。本研究将每个样本的约2000万条序列读段比对至小鼠基因组，在野生型与TLR4ΔIEC小鼠的回肠组织中共鉴定到86477个基因。其中455个基因在两组小鼠间存在差异表达，另有1037个基因仅在野生型或TLR4ΔIEC小鼠的单一组内存在表达，本次分析采用的G因子阈值为0.015，差异倍数截断值为0.54。通过IPA进行通路分析显示，部分受影响的经典KEGG通路包括DNA复制、过氧化物酶体增殖物激活受体（PPAR）信号通路、Toll样受体信号通路、细胞因子-细胞因子相互作用、黏着连接、Jak-STAT信号通路以及紧密连接。研究人员从3只野生型小鼠与3只TLR4ΔIEC小鼠的回肠中提取总RNA，并在全转录组文库构建过程中，为每只个体小鼠的样本添加了特异性条形码。随后将3只野生型小鼠（条形码BC01、BC02、BC03）的混合样本与3只TLR4ΔIEC小鼠（条形码BC07、BC08、BC09）的混合样本分别进行RNA测序。