title: RNA-seq of human liver biopsies from patients with NAFLD, patient with cirrhosis, and healthy controls under both fasting and postprandial conditions.
design: RNA-seq of human liver biopsies from patients with NAFLD, patient with cirrhosis, and healthy controls under both fasting and postprandial conditions.
library_construction_protocol: A balloon catheter was placed in the vena jugularis interna under local anesthesia and the correct position was controlled using sonography and x-ray. The catheter was moved forward and placed in the liver vein. A liver biopsy (19-G Tru-cut) was taken with a needle punctures. The whole procedure was done under sterile conditions by experienced specialists. In all subjects, histological assessment was performed on a second liver tissue sample. The liver biopsy sample was stored at -80 ◦C and transported with the use of dry ice to ensure optimal stability of the tissue samples. Healthy controls consumed a standardized liquid meal between 6PM and 10PM the night before the study day, consisting of two Nutridrink Standard (Nutricia, 200 ml) containing 300 kcal (16% protein, 49% carbohydrates and 35% fat) each. All participants met fasting at 8AM (fasting since 10PM). Participants randomised to receiving a standardized meal (postprandial) drank a standardized fluid meal, Nutridrink (Nutridrink Standard, 200 mL, 300 kcal, Nutricia) over 5 minutes 60 minutes prior to the liver biopsy. Participants randomised to fasting stayed fasting. Raw sequencing data were processed using bcl2fastq software version 2.20.0 (Illumina). Total DNA/RNA was purified by an AllPrep DNA/RNA Minikit (cat 80204 - QIAgen) according to manufacturer's instructions and qualified using a 2100 Bioanalyzer instrument (Agilent Genomics). The extracted DNA/RNA were DNase treated with RNeasy Minikit (cat 74106 - QIAgen) and Rnase Free DNase set (cat 79254 - QIAgen) according to manufacturer's protocol. All the above-mentioned processes was performed on a QiaCube machine. Library preparation was done using TruSeq Stranded Total RNA Library Prep Gold (cat 20020599 - Illumina) and IDT for Illumina-TruSeq RNA UD Indexes (cat 20022371 - Illumina) according to the manufacturer's instructions. Some samples were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat NEB-E7760S - New England Biolabs), NEBNext rRNA Depletion Kit 24 with beads (cat NEB-E7765 - New England Biolabs), and NEBNext Multiplex Oligos for Illumina (cat E7600S - New England Biolabs).