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ERX4742573: Illumina HiSeq 4000 sequencing; Liver transcriptome of biopsy-proven NAFLD patients at different stages of the disease
1 ILLUMINA (Illumina HiSeq 4000) run: 8.8M spots, 438.3M bases, 186.1Mb downloads

Design: Liver transcriptome of biopsy-proven NAFLD patients at different stages of the disease
Submitted by: Co-author
Study: Liver transcriptome of biopsy-proven NAFLD patients at different stages of the disease
show Abstracthide Abstract
We investigated the hepatic transcriptome of 58 biopsy-proven NAFLD patients at multiple stages of the disease (NAFL, NASH with mild fibrosis, NASH with advanced fibrosis) with the aim of describing the pathophysiological events driving the development and progression of NASH.
Sample: Sample 8
SAMEA7625706 • ERS5383993 • All experiments • All runs
Organism: Homo sapiens
Name: Sample 8_s
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
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
Experiment attributes:
Experimental Factor: disease: non-alcoholic steatohepatitis
Experimental Factor: disease staging: NASH Fibrosis Stage 3-4
Runs: 1 run, 8.8M spots, 438.3M bases, 186.1Mb
Run# of Spots# of BasesSizePublished


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