DOI: 10.7280/d1vx0c
title: Differentially expressed genes in the blood, spleen, and liver of Peromyscus leucopus and Mus musculus with or without treatment with lipopolysaccharide

description: Animals that are competent reservoirs of zoonotic pathogens commonly
 suffer little morbidity from the infections. To investigate mechanisms of
 this tolerance of infection, we used single-dose lipopolysaccharide (LPS)
 as an experimental model of inflammation and compared the responses by two
 rodents: Peromyscus leucopus, white-footed deermouse and reservoir for
 agents of Lyme disease and other zoonoses, and the house mouse Mus
 musculus. Four hours after injection with LPS or saline, blood, spleen and
 liver samples were collected and subjected to RNA-seq, metabolomics, and
 specific RT-qPCR. Differential expression analysis was at gene, pathway,
 and network levels. LPS-treated deermice showed similar signs of sickness
 as exposed mice and had similar increases in corticosterone levels and
 expression of interleukin (IL)-6, tumor necrosis factor, IL-1beta, and
 C-reactive protein. By network analysis the M. musculus response to LPS
 was characterized as cytokine-associated, while the P. leucopus response
 was dominated by neutrophil activity terms. In addition, dichotomies in
 expression of arginase 1 and nitric oxide synthase 2 and of IL-10 and
 IL-12 were consistent with type M1 macrophage responses in mice and type
 M2 in deermice. This dataset comprises the list of differentially
 expressed genes from the RNA-seq analysis of 20 P. leucopus (10 females
 and 10 males) and 20 M. musculus (10 females and 10 males) that were
 inoculated intraperitoneally with 10 mg/kg of E. coli lipopolysaccharide
 (6 females and 6 males of each species) or with saline alone (4 females or
 4 males of each species) at time 0 and then the animals were euthanized at
 4 hours. The tissues were then processed for RNA extraction, cDNA library
 preparations, and sequencing and then analysis as described under
 Methods. 
description: Animals.  Adult outbred P. leucopus of the LL stock were obtained
 from the Peromyscus Genetic Stock Center (PGSC) of the University of South
 Carolina (101). The LL stock colony was founded with 38 animals captured
 near Linville, NC between 1982 and 1985 and has been closed since
 1985. Animals were maintained in the AAALAC-accredited U.C. Irvine
 vivarium with 2-5 animals per cage according to sex and on 12 hours
 light-12 hours dark lighting schedule, temperature of 21-23° C, humidity
 of 30-70%, water ad libitum, and a diet of 8604 Teklad Rodent (Harlan
 Laboratories).  Single dose LPS comparison. Animals
 were anesthetized with isoflurane and injected i.p. with a single dose of
 E. coli O111:B4 LPS at a concentration of 10 mg/kg body weight in a 50 µl
 volume as described above. The control group was anesthetized and then
 injected with the 0.9% saline alone. The experiment started at 0800 h with
 10 min intervals between animals and with alteration of LPS and control
 injections. At 4.0 hr after their injection the animals were euthanized.
 After opening the chest, exsanguination was performed by cardiac puncture
 and blood was transferred to a heparin sulfate coated tube
 (Becton-Dickinson Microtainer). Anticoagulated blood was centrifuged to
 pellet blood cells for 3 min at 4600 x g at 4° C. Plasma and blood pellet
 was kept separately at -80° C until further analysis. Liver and spleen
 were extracted, flash-frozen in liquid nitrogen, and stored at -80 °C
 until RNA extraction.  RNA extractions. RNA from blood
 was extracted using NucleoSpin RNA Blood Mini kit (Macherey-Nagel). Lysis
 buffer and proteinase K were added to the frozen pellet and shaken while
 thawing. RNA from liver and spleen was extracted from 30 mg of frozen
 tissues, mechanically homogenized and further lysed in RLT buffer with
 2-mercaptoethanol on TissueLyser (Qiagen) with 3 mm beads then extracted
 according to the protocol using RNeasy Mini Kit (Qiagen).
 RNA-seq. Library preparation with the Illumina TruSeq mRNA
 stranded kit was carried out. The libraries were normalized and then
 multiplexed to achieve 12 samples per flow cell on an Illumina HiSeq 4000
 instrument and 100 or 150 cycles of paired-end read chemistry at the U.C.
 Irvine Genomic High Throughput Facility. The quality of sequencing reads
 was analyzed using FastQC (Babraham Bioinformatics). The reads were
 trimmed of low-quality reads (Phred score of &lt;15) and adapter
 sequences, and corrected for poor-quality bases using Trimmomatic. After
 these steps there were overall for all the studies between 45 to 100
 million reads for each of the samples. For the comparative study of 40
 animals the mean and median numbers, respectively, of reads (x 10^6) for
 the three tissues were 62.9 and 62.9 for blood, 65.2 and 63.8 for spleen,
 and 60.5 and 59.4 for liver. Blood and liver RNA-seq were PE100, and
 spleen RNA-seq was PE150. Paired-end reads in fastq files were quantified
 using kallisto v. 0.46.1, using the GENCODE annotation v. 21
 (https://www.gencodegenes.org) for mouse and GCF_004664715.1_Pero_o.1 _rna
 from NCBI for P. leucopus. Differential expression. We
 used edgeR v. 3.28.1 for differential gene expression (DEG) analysis.
 Genes were called differentially expressed if their absolute fold-change
 between conditions was &gt; 4.0 and the false-discovery rate (FDR) was
 &lt;0.05. To compare fold-changes across species, we merged the output
 tables from the DEG analyses and retained 14,685 orthologous genes that
 were synonymously annotated between both species, out of total 24,295
 annotated genes for P. leucopus and 35,805 for M. musculus. To screen for
 DEGs that varied in magnitude by species, we required an absolute fold
 change of &gt; 5.0 (log2 = 2.5) in one species and &lt; 5.0 in the
 other. For DEGs designated as "shared" between the species, the
 absolute fold change was &gt; 5.0 in both. The
 BioProject for this experiment is PRJNA643535. The BioSamples numbers for
 the samples for this experiment are SAMN15445905-SAMN15445964. SRA archive
 accession numbers for the fastq files of PE100 and PE150 reads
 are SRR12782328-SRR12782387.
description: The data is a single spreadsheet entitled "Table of
 differentially expressed genes (DEG) of Peromyscus and Mus musculus in
 response to lipopolysaccharide".  The data are in 6 groups, each
 occupying four columns from the left to right: DEGs of the blood of P.
 leucopus, DEGs of the spleen of P. leucopus, DEGs of liver of P. leucopus,
 DEGs of blood of M. musculus, DEGs of spleen of M. musculus, and DEGs of
 liver of M. musculus. The four columns in each group are ascending rank by
 false-discovery rate (FDR) p value, gene or locus name, log2 value of the
 fold-change of LPS-treated over control animals, and FDR.