Differentially expressed genes of Peromyscus leucopus fibroblast cultures treated with lipopolysaccharide or buffer alone

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.