Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and PPARbeta-null mice during liver regeneration
Purpose: The current study tests the hypothesis that peroxisome proliferator-activated receptor ß (PPARß) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. Methods: Wild-type (WT) and PPARß-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period. Standard PH was performed. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the University of California Davis Animal Care and Use Committee.Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 µg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads. Results: Differential gene expression profiling revealed the inhibition of expression in genes and pathways that are involved in metabolism and proliferation in regenerating KO livers. Specifically, PPARß deficiency affected the activation of Akt and the expression of E2fs. Pathways that control glycolysis, FA synthesis as well as cell proliferation were de-regulated in regenerating PPARß KO livers. Conclusions: The data suggest a role for PPARß in regulating liver regeneration that is mediated at least in part through Akt and E2f-regulated pathways. Overall design: mRNA profiles of 3-month old wild type (WT) and PPARbeta mice were generated by deep sequencing, one simple, using Illumina HiSeq 2000 instrument with 100 bp paired end reads