of age, whole body fat, fluids and lean tissue mass of mice were determined using a Bruker Optics Minispec nuclear magnetic resonance (NMR) analyzer (The Woodlands, TX, USA) according to the manufacturer’s recommendations. We also calculated the total mass of the mice, sum of lean mass, free fluid, and fat mass, and body fat percentage, fat mass/total mass. Following a 16-hour fast, mice were weighed and then bled retro-orbitally under isoflurane anaesthesia. Complete blood counts were performed using a Heska CBC-Diff analyzer (Heska Corp, Loveland, CO, USA). An external control sample with known analyte concentra- tion was run in each plate to ensure accuracy. Glucose levels were determined using commercially available kits from Sigma (St Louis, MO, USA). Insulin levels were measured using commercial ELISA kits (ALPCO Diagnostics). All measurements were performed in triplicate according to the manufacturer’s instruc- tions. Plasma lipids were determined as previously described [35]. Mice were euthanized by cervical dislocation and the mass of individual tissues and fat depots (heart, kidney, retroperitoneal fat pad, epididymal fat pad, subcutaneous fat pad, and omental fat pad) were determined by dissecting and weighing each tissue/pad separately after the mice were euthanized. Following this, liver tissues were dissected out, flash frozen in liquid nitrogen, and kept at 270 degrees until further processing. RNA isolation, expression profiling, and RNA-Seq experiment At 16 weeks of age, the liver tissues of the mice were dissected out, flash frozen in liquid nitrogen, and kept at 270 degrees until further processing. For RNA profiling the RNA from 3 mice per strain were hybridized to Affymetrix Mouse Genome HT_MG- 430A arrays. Frozen liver samples were weighed and homogenized in Qiazol according to the manufacturer’s protocol. Following homogenization, RNA extraction was performed using Qiagen’s RNeasy kit (cat# 74104). Ninety two strains of mice had three biological replicates, five strains had two biological replicates and two strains had one biological replicate each. All RNA samples were cleaned using a Biosprint96 (Qiagen, Valencia, CA) with RNA cleanup beads (Agencourt Bioscience, Beverly, MA) following manufacturer’s protocol with adaptations for use with the Biosprint. The quality of the total RNA from the samples was monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and RNA quantity was measured with a NanoDrop (NanoDrop Technologies, Inc. Wilmington, DE) following the manufacturer’s instructions. All samples were arrayed into three 96 well microtiter plates following a randomized design format that places samples from the same strain on different plates to better estimate variance across testing strains. All target labeling reagents were purchased from Affymetrix (Santa Clara, CA). Double-stranded cDNAs were synthesized from 1 ug total RNA through reverse transcription with an oligo-dT primer containing the T7 RNA polymerase promoter and double strand conversion using the cDNA Synthesis System. Biotin-labeled cRNA was generated from the cDNA and used to probe Affymetrix Mouse Genome HT_MG-430A arrays. The HT_MG-430A Array plate consists of 96 single MG-430A arrays arranged into standard SBS 96 well plate format. All cDNA and cRNA target preparation steps were processed on a Caliper GeneChip Array Station from Affymetrix. Array hybridization, washing and scanning were performed according to the manu- facturer’s recommendations. Scanned images were subjected to visual inspection and a chip quality report was generated by the Affymetrix’s GeneChipOperating System (GCOS) and Expression console Affymetrix). Two of 288 chips were excluded due to low QC scores. The image data was processed using the Affymetrix Global Mouse mRNA and Protein Levels Comparison GCOS algorithm utilizing quantile normalization or the Robust Multiarray method (RMA) to determine the specific hybridizing signal for each gene. Expression data can be obtained from Geo database (GSE16780). To avoid the effect of SNP on hybridiza- tion, we matched the location of ,14 million SNPs from dbSNP database (NCBI) to the location of the individual probes on the genome. If the location of the probe had a matching SNP within it we flagged the probe and exclude it from the cdf file prior to RMA normalization. If a probeset contained SNP in 8 or more 25-mer probes, we excluded the probeset from the analysis. The cleaned datasets were then background corrected and normalized using the affy package (from bioconductor) using rma, pmonly, and median-polish normalization methods. RNA isolation for Next Generation Sequencing followed the same protocol as the one described above for the microarray data. For the RNA-Seq experiment, two inbred mice (C57BL/6J and DBA/2J) were chosen. Library preparation was carried using illumina’s mRNA-Seq 8-Sample Prep Kit protocol (Illumina, cat# RS-100-0801). In brief, 1 to 10 ug of RNA was used for library construction. In the first step the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Next, the purified mRNA was fragmented into small pieces using divalent cations, followed by double stranded cDNA synthesis using random primers, adenylated at the 39 end and ligated to the sequencing adapters. The ligated products were then separated on 2% agarose gel, 200 bp fragments were selected and PCR amplified using PE 1.0 and PE 2.0, and purified using QIAquick PCR Purification Kit (QIAGEN, part # 28104). The final library concentration was verified by Bioanalyzer. Sequenc- ing reaction was performed by Illumina Genome Analyzer 2.0 at UCLA Human Genetics microarray core. Raw sequences were uploaded onto Galaxy website (at http://main.g2.bx.psu.edu/) and using the Tophat software [22] was aligned against the reference genome (M. musculus, mm9) downloaded from UCSC. Alignment was performed by setting the parameter for misalign- ment to one. Relative abundance of transcripts (in Fragments Per Kilobase of Exon per Million read sequence units) was estimated using the Cufflink software [23] and the Ensembl’s Mus_musculus NCBIM37 as the reference annotation file. Protein isolation and sample preparation Male mice were euthanized using isoflurane followed by cervical dislocation at 6–10 weeks of age. The liver tissue was immediately frozen in dry ice until further processing. The 97 samples corresponding to different mouse strains plus some extra samples from C57BL/6J mouse were randomized into 10 batches of 10 samples. Each batch was processed separately prior to quantitative LC-MS analysis. Before the LC-MS analysis the batches were put together and the sample list was randomized one more time. The extraction and digestion of the proteins was performed using a commonly used protocol based on denaturation of protein in 8 M urea followed by digestion with trypsin. Briefly, approximately 5 mg of liver tissue was resuspended in 100 ul denaturing solution (8 M urea, 50 mM Tris-HCl pH 8.0 and 1 mM EDTA) and homogenized with a motorized pestle. Upon homogenization, the total protein content was measured by Bicinchoninic Protein Assay (BCA, Pierce, Rockford, IL) and the 500 ug aliquots were taken from each sample for further processing. DTT was added to a concentration of 10 mM in sample, then to solubilize and unfold the proteins the samples were incubated for 30 min at 37oC with shaking. Cysteine residues were alkylated by adding iodoaceta- mide up to 40 mM concentration and incubating for 1 hour at 37oC, with shaking, in the dark. For protein digestion the samples were diluted 10-fold with 50 mM ammonium bicarbonate PLoS Genetics | www.plosgenetics.org 13 June 2011 | Volume 7 | Issue 6 | e1001393