2.2. RNA Sequencing of Human Tissue NAFLD

For human RNAseq studies, human liver samples were obtained from the Human Biorepository Core from the NIH-funded international InTeam consortium (7U01AA021908-05) as previously described [36]. All patients gave written informed consent and the research protocols were approved by the local Ethics Committees and by the central Institutional Review Board of the University of North Carolina at Chapel Hill. For the present study, we compared non-diseased normal human livers (N = 10) with NAFLD patients according to Kleiner’s Criteria and without alcohol abuse (N = 9) (Supplementary Table S3). Patients with malignancies were excluded from the study.

RNA extraction, sequencing, and bioinformatic analysis: RNA extraction and sequencing was performed as indicated previously [36]. Total RNA from flash-frozen liver tissue was extracted by phenol/chloroform separation (TRIzol, Thermoscientific, Waltham, MA, USA). RNA purity and quality were assessed by automated electrophoresis (Bioanalyzer, Agilent, Santa Clara, CA, USA) and sequenced using Illumina HiSeq2000 platform. Libraries were built using TruSeq Stranded Total RNA Ribo-Zero GOLD (Illumina, San Diego, California, USA). Sequencing was paired end (2 × 100 bp) and multiplexed. Ninety-four paired-end sequenced samples obtained an average of 36.9 million total reads with 32.5 million (88%) mapped to GRCh37/hg19 human reference. Short read alignment was performed using STAR alignment algorithm with default parameters. To quantify expression from transcriptome mappings we employed RSEM.