DOI: 10.7280/d1md69
title: Differentially expressed genes of Peromyscus leucopus fibroblast cultures treated with lipopolysaccharide or buffer alone

description: This experiment and its results were part of a larger study of the
 differential responses of the white-footed deermouse Peromyscus leucopus
 and the house mouse Mus musculus to single injections
 of lipopolysaccharide (LPS). The entire study is
 presented in the associated manuscript in submission and in the
 listed pre-print. The aim of this particular experiment was to identify
 differentially expressed genes (DEG) of fibroblast cultures of P.
 leucopus. The experiment identified 543 DEGs that were either upregulated
 or down-regulated in response to four hours' exposure to LPS. The
 accompanying dataset identifies these DEGs and gives the fold-change of
 LPS-treated over control samples as well as the false-discovery rate (FDR)
 p value. For protein coding sequences the 10 highest TPMs in order among
 control samples were ferritin heavy chain (Fth), Slpi, secreted protein
 acidic and cysteine rich (Sparc), eukaryotic translation elongation factor
 1 alpha (Eef1a1), ferritin light chain (Ftl), vimentin (Vim), serpin
 family H member 1 (Serpinh1), ribosomal protein lateral stalk subunit P1
 (Rplp1), collagen type I alpha 1 chain (Col1a1), and Gapdh. There were 324
 genes that were up-regulated by criterion of fold-change ≥ 4 and FDR
 &lt; 0.05, and 17 genes that were down-regulated, with an additional
 80 down-regulated by criterion of fold-change ≥ 2 and FDR &lt; 0.5.
 Among those displaying the marked increases in expression between the
 control and LPS conditions were two subunits of nuclear factor kappa B
 (Nfkb), two forms of Saa, Nos2, Csf3, Sod2, and Pla2g2a.
description: Fresh ear punches were collected from five LL stock P. leucopus
 animals (2 females and 3 males) during routine marking procedures at the
 time of weaning at ~3 weeks of age. After the ear punch tissue was bathed
 in 70% ethanol for 2 min, it was placed in RPMI 1640 medium (HyClone
 FetalClone II; Thermo Scientific) supplemented with 10% fetal bovine serum
 (Gibco), 100 U/ml penicillin, 100 mg/ml streptomycin and 0.292 mg/ml
 L-glutamine (HyClone). Ear punches were minced, and then treated with 2
 mg/ml collagenase type I (Millipore) for 1 h. Undigested debris was
 removed once cells were visible.  The disassociated cells were cultivated
 in the same medium at 37° C and in 5% CO2. Cells were passed when adherent
 layers reached 90% confluency and for no more than 7 passages. For the
 experiment individual cultures were split into pairs and they were
 incubated at initial concentrations of 3 x 105 cells per well for 24 h. E.
 coli O111:B4 LPS or saline alone was added medium for final LPS
 concentration of 1 µg/ml and then the incubation was continued for 4 h.
 After disassociation of the fibroblast layer with trypsin and then
 addition of RNAlater (Thermo Scientific), the cells were harvested and
 stored at a concentration of ~10^6/ml in -80 ℃ until RNA extraction with
 RNeasy Mini Kit (Qiagen) after homogenization.  cDNA
 libraries were prepared with the Illumina TruSeq mRNA stranded kit. The
 libraries were normalized and then multiplexed to achieve 12 samples per
 flow cell on an Illumina HiSeq 4000 instrument and 100 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. The transcript reference
 set GCF_004664715.1_Pero_o.1 _rna from NCBI for P. leucopus. CLC Genomics
 Workbench v. 20 (Qiagen) was used alignments of reads to the reference
 sets and for differential gene expression (DEG) analysis. Of the 46,141
 transcripts in the reference set, 18,462 had a mean TPM of &gt; 1 in
 either the control or LPS group, and these were used the DEG
 analysis.
description: The dataset is a single Excel spreadsheet entitled "Table.
 Differentially expressed genes of Peromyscus leucopus fibroblast cultures
 treated with lipopolysaccharide or buffer alone". The variables in
 columns are from the left accession number for P. leucopus transcript,
 gene name, gene product name, maximum (max) group mean expression level in
 TPM, fold-change, and negative log10 of the false discovery rate (FDR) p
 value. The rows are sorted by descending order of fold-change of
 LPS-treated to control members of a fibroblast culture pair.  A README
 file is included with further details.  The BioProject
 identification for this experiment is PRJNA672217.  The BioSample
 accession numbers for the 5 pairs (total of 10) samples are SAMN16563388-
 SAMN16563397.  The SRA accession numbers for the fastq files of the
 Illumina sequencing reads for the RNA-seq
 are SRR13021354-SRR13021363.