organism: Homo sapiens
description: Protocols: Primary human hepatocytes were isolated from patients undergoing liver resection, from extirpated livers or from donor livers rejected for transplantation. The isolation procedure followed a three-step perfusion technique by connection of one or two catheters to the liver vessels. The liver was first perfused with buffer containing EGTA to break up desmosomes between cells. Following a wash out with buffer, the liver tissue was digested with collagenase. The liver capsule was cut to release cells and the cells were washed three times by low-speed centrifugation. Cells were cultured on matrigel derived from Engelbreth-Holm-Swarm sarcoma (Sigma-Aldrich, St. Louis, MO) and in William's E medium (Invitrogen, Waltham, MA) supplemented with 20 mM HEPES (Lonza, Basel, Switzerland), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO), 10 nM insulin, 100 nM dexamethasone, 0.01 M gentamicin (Lonza, Basel, Switzerland) and 0.055 µM amphotericin B, for five days at 37C in 5% CO2. Cells were lysed in TRIzol (Invitrogen, Waltham, MA) and kept at -80C until further processing. Primary human hepatocytes were kept in culture for a total of 5 days prior to harvesting and reagents were added at concentrations and time points for the different experiments as follows: For the recombinant experiment, cells were treated with 10 M CDCA (Sigma-Aldrich, St. Louis, MO) or 1 ng/mL recombinant FGF19 (R&D systems, Minneapolis, MN) for 6 h (n=3). For the endogenous experiment (n=3), cells were treated with 40 M CDCA (induction medium), to induce production of FGF19, or regular medium without CDCA (control medium) for 6 h. Cells were washed, and medium changed, and cells were kept for an additional 18 h (condition/control medium), during which the cells continued to produce FGF19. Conditioned or control medium was then transferred to untreated cells that were maintained for 24h. RNA was extracted using TRIzol-based phase separation (Invitrogen, Waltham, MA) according to manufacturer's instructions. The library preparation was carried out using the Illumina TruSeq® Stranded RNA Library Prep Kit v2 (dual index) according to the manufacturer's instructions. Ribosomal RNA depletion was achieved with Ribo-Zero Gold. The quality of every cDNA library was determined on an Agilent Bioanalyzer instrument according to the manufacturer's protocol. All libraries were quantified with the KAPA-SYBR FAST qPCR kit (Roche).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-01-16T04:12:41Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-12-20T10:49:20Z
attributes.tag: External Id
attributes.value: SAMEA6432151
attributes.tag: INSDC center name
attributes.value: Department of Microbiology, Tumor, and Cell Biology, Karolinska Institutet, Science for Life Laboratory, Sweden
attributes.tag: INSDC first public
attributes.value: 2021-01-16T04:12:41Z
attributes.tag: INSDC last update
attributes.value: 2019-12-20T10:49:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8627:Sample 11
attributes.tag: age
attributes.value: 31
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: hepatocyte
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: individual
attributes.value: VF39
attributes.tag: organism part
attributes.value: liver
attributes.tag: replicate
attributes.value: 3
attributes.tag: sample name
attributes.value: E-MTAB-8627:Sample 11
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male