Differentially expressed genes of Peromyscus leucopus fibroblast cultures treated with lipopolysaccharide or buffer alone
Barbour, Alan; Zhang, Youwen; Milovic, Ana; Kiaris, Hippokratis (2021), Differentially expressed genes of Peromyscus leucopus fibroblast cultures treated with lipopolysaccharide or buffer alone, Dryad, Dataset, https://doi.org/10.7280/D1MD69
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 < 0.05, and 17 genes that were down-regulated, with an additional 80 down-regulated by criterion of fold-change ≥ 2 and FDR < 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.
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 <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 > 1 in either the control or LPS group, and these were used the DEG analysis.
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.
National Institutes of Health, Award: AI-136523
National Science Foundation, Award: OIA-1736150