Name: Sample 52_s
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: Oligo-dT
Layout: SINGLE
Construction protocol: Our “BioNASH” Cohort consisted of 58 consecutive patients recruited by the NAFLD Service at Cambridge University Hospitals NHS Foundation Trust. This study was approved by the local Ethics Committee (Reference number: 06/Q0106/70). All patients gave their informed consent for the use of data (biochemistry and clinical history) and samples for research purposes; the principles of the Declaration of Helsinki were followed. All the patients had a clinical diagnosis of NAFLD (patients with alternate diagnoses and etiologies were excluded), histology scored by an expert pathologist, and snap-frozen tissue for research purposes. Human biopsies' RNA was isolated using STAT-60 (AMS biotechnology, CS-502) according to the following procedure: 1) biopsies were homogenised in STAT-60 (1 ml) using a tissue homogeniser, mixed (vortexing) and centrifuged at 13,000 g for 5 minutes at RT; 2) the supernatant was mixed (vortexing) with 200 ml chloroform (Sigma, Cat 650471) and centrifuged (12,000g) for 15 minutes at 4°C; 3) the supernatant was then mixed with 500 ml isopropanol (Sigma, cat 33539) and centrifuged at 10,000g for 10 minutes at 4°C to pellet the RNA; 4) the pellet was washed with 75% ethanol (1 ml) and allowed to dry until evaporation; 5) RNA was re-suspended in RNAse free water (Thermo Fisher Scientific, Delaware USA). All reagents, plastic ware, and supplies used were nuclease-free, sterile, and of molecular biology grade. RNA purity (A260/A280>1.80) and concentration were determined using the Nanodrop spectrophotometer (Thermo Fisher Scientific, Delaware USA). RNA integrity was studied using the 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kits (Agilent, Santa Clara, California, USA). RNA Integrity Number (RIN) of 7 was considered the lowest cut-off for RNA sequencing. RNA from tissues (600 ng RNA) were used to generate barcoded sequencing libraries using Illumina TruSeq® Stranded mRNA Library Preparation Kit (Illumina) following manufacturer's instructions. Library preparation was performed by the Genomics and Transcriptomic Core at the Institute of Metabolic Science