Differential gene expression analysis for BAL cells isolated from LysM Cre and Netrin1loxp/loxp LysM Cre mice after intratracheal LPS challenge

Purpose: The goal of this study is to compare NGS-derived transcriptome profiling (mRNA-seq) of expressed genes between LysM Cre and Netrin1loxp/loxp LysM Cre mice 3 days after intratracheal LPS challenge in order to explain the worsened outcomes and increased levels of inflammation we measure in the Netrin1loxp/loxp LysM Cre mice Methods: Three days after mice were treated with intratracheal injections of LPS, BAL cells were collected and then depleted for neutrophils using antibody-mediated depletion. The remaining cells were used for RNA isolation. mRNA profiles were generated by deep sequencing, in quadruplicate (one sample in the Netrin1loxp/loxp LysM Cre group was excluded as the sequence depth was only half compare to other 3 replicates), using paired-end 75-cycle sequencing on an Illumina NextSeq 550 System. Bases with quality scores < 20 and adapter sequences were removed from raw data with Cutadapt (v1.15), followed by alignment of clean RNA-seq reads to GRCm38 with STAR(v2.5.3a). Gene abundance was counted by HTseq-count uniquely-mapped reads number with default parameter using GencodeM15. Genes with > 5 reads in at least one sample were included for differential expression analysis by DESeq2 software. DEG analysis from LysM Cre and Netrin1loxp/loxp LysM Cre mice after intratracheal LPS challenge

研究目的：本研究旨在通过下一代测序（Next-Generation Sequencing, NGS）技术获取表达基因的转录组谱（mRNA测序，mRNA-seq），并对比气管内脂多糖（Lipopolysaccharide, LPS）刺激3天后的LysM Cre与Netrin1loxp/loxp LysM Cre小鼠的转录组差异，以阐释Netrin1loxp/loxp LysM Cre小鼠中观测到的不良结局与炎症水平升高的潜在机制。
实验方法：小鼠经气管内注射脂多糖3天后，收集支气管肺泡灌洗液（Bronchoalveolar Lavage, BAL）细胞，通过抗体介导法完成中性粒细胞耗竭处理，剩余细胞用于RNA提取。随后采用Illumina NextSeq 550测序系统（Illumina NextSeq 550 System）进行双端75循环测序，以四重生物学重复开展实验（Netrin1loxp/loxp LysM Cre组中有1个样本因测序深度仅为其余3个样本的一半而被排除），生成mRNA表达谱。原始测序数据经Cutadapt（v1.15）去除质量值低于20的碱基与接头序列，将过滤得到的clean reads通过STAR(v2.5.3a)比对至小鼠参考基因组GRCm38。使用HTseq-count结合GencodeM15注释文件，以默认参数统计唯一比对reads的基因丰度。仅保留至少1个样本中reads数大于5的基因，通过DESeq2软件完成差异表达基因（Differentially Expressed Gene, DEG）分析，该分析针对气管内脂多糖刺激后的LysM Cre与Netrin1loxp/loxp LysM Cre小鼠进行。