LPS/NKA signaling study in macrophages-Bulk RNA Sequencing data

Published: 8 August 2022| Version 1 | DOI: 10.17632/vxyjg9m7nm.1
Contributor:
Yiliang Chen

Description

We hypothesize that lipopolysaccharides (LPS) stimulate higher pro-inflammatory responses in murine peritoneal macrophages with reduced Na/K-ATPase (NKA) alpha1 expression. We conducted bulk RNA sequencing on WT and atp1a1 heterozygous null macrophages treated with control (cell culture media), or LPS 100ng/ml 6h (n=3). We attach here the raw read count as well as normalized read count on all genes from the RNA sequencing.

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WT and atp1a1 heterozygous null macrophages treated with control (cell culture media: RPMI1640 plus 1% Penicillin/Streptomycin), or LPS 100ng/ml in cell culture media 6h (n=3). Total RNA were isolated by RNA isolation kit (Qiagen) and subjected to bulk RNA Sequencing performed by the company MedGenome Inc. The Mouse samples sequencing was done using Illumina HiSeqX platform. SMARTer® Stranded Total RNA Library Prep Kit was used to perform RNA sequencing. The raw reads are aligned using STAR (2.4.1) aligner.Reads mapping to ribosomal and mitochondrial genome were removed before performing alignment. Overall more than 90% of the total pre-processed reads mapped to the reference gene model/genome. The raw read counts were estimated using HTSeq-0.6.1. Read count data were normalized using DESeq2. Hierarchical clustering analysis and PCA analysis is performed for normalized counts of all the protein coding genes for all the samples. Euclidean distance and the complete linkage clustering method is used for hierarchical clustering. Analysis is performed using the R language and additional packages: ggplot2, reshape2 and ggrepel. Differential expression analysis is performed using DESeq2 (R Bioconductor package). Significant differential expression cutoff: P value < 0.05 and Fold change >=2 or <=-2. Pathway analysis was conducted using “gseKEGG” method in “clusterProfiler” package to generate the differential pathway. GSEA plot was plotted by function “gseaplot2” in enrich plot library under bioconductor package.

Institutions

Medical College of Wisconsin

Categories

RNA Sequencing

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