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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE167333 | GEO | 2021/02/24

REPOSITORIES: GEO

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

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

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2014-07-11 | E-GEOD-59319 | biostudies-arrayexpress

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Project description:Purpose: The goals of this study are to compare genes change between FBXW7f/f and LysM-cre FBXW7f/f BMDM after LPS treatment Methods: BMDM mRNA from 6-8weeks FBXW7f/f (WT) and LysM-cre FBXW7f/f (KO) mice were generated by transcription sequencing, using Illumina. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.

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RNA-seq analysis of Mettl3fl/fl and LysM-cre, Mettl3fl/fl BMDMs (bone-marrow-derived macrophages) Transcriptomes

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Transcription profiling by array of LysMCre/Cre and KLF2?/? (LysMCre/Cre: KLF2 FL/FL) primary peritoneal macrophages treated with lipopolysaccharide (LPS) for 6 hours

Project description:Gene expression profile of LysMCre/Cre and KLF2?/? primary peritoneal macrophages following 6 hours of LPS treatment. We used microarrays to detail the global program of gene expression following LPS stimulation of LysMCre/Cre and KLF2?/? primary peritoneal macrophages. We identified distinct classes of genes that were altered following LPS stimulation. We injected 3 pairs of LysMCre/Cre (n=3) and KLF2?/? (n=3) mice with 2 ml of thioglycolate broth intraperitoneally. After 72 hours of thioglycolate injection, total cell suspension from peritoneal cavity were collected. These cells were seeded on 100mm tissue culture plates. Cells were allowed to attach for 4 hours and unattached cells were removed by washing with cell culture medium. These cells were placed in humidified incubator for additional 24 hours before LPS treatment. LPS was diluted to final concentration of 100ng/ml in culture medium and this medium was placed on peritoneal macrophages derived from LysM Cre/Cre and KLF2?/? mice for 6hours. Cells were lysed and total RNA was isolated and subjected to hybridization on Affymetrix microarrays.

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