RNA-seq analysis of Ehrlichia muris infection induced murine splenic and hepatic memory B cells subsets.

Purpose: The goal of this study is to complare the transcitpional profiles of freshly isolated, murine splenic and hepatic memory B cells (B220+ CD73+) sorted as CD11b, CD11c double negative and CD11b, CD11c double positive cell populations induced by Ehrlichia muris infection. Methods: For mRNA sequencing analysis, B220 positive, CD73 positive MBCs were sorted as CD11b, CD11c double negative and CD11b, CD11c double positive. After the sort, the cells were spun down and washed with PBS and re-suspended in RLT+1% beta-mercaptoethanol. RNA was prepared with RNAeasy microplus kits from Qiagen, according to recommended protocol. cDNA libraries were constructed using SMARTer low input kit for mRNA seq. Samples were sequenced using Illumina NextSeq 500 with 75 bp paired-end reads and aligned to the mm10 genome using the STAR aligner ((Dobin et al., 2013). The number of uniquely aligned reads ranged from 22 to 32 million. Gene-level counts were determined using featureCounts (Liao et al., 2014), and raw counts were quantile normalized to each other for differential expression using the voom method (Law et al., 2014) in the limma R package (Ritchie et al., 2015) The presence of certain liver-specific transcripts indicated unavoidable liver cell contamination in the liver sample; to prevent this from confounding our analysis, differential expression was performed only on genes, which were having at least 30 counts in the all liver and spleen samples. All gene-set enrichments were preformed using the rankSumTestWithCorrelation function in limma, which explicitly corrects for correlation among genes in the gene set being interrogated.