Sample 25
Homo sapiens
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).
ENA-FIRST-PUBLIC
2021-01-16T04:12:41Z
ENA-LAST-UPDATE
2019-12-20T10:49:20Z
External Id
SAMEA6432157
INSDC center name
Department of Microbiology, Tumor, and Cell Biology, Karolinska Institutet, Science for Life Laboratory, Sweden
INSDC first public
2021-01-16T04:12:41Z
INSDC last update
2019-12-20T10:49:20Z
INSDC status
public
Submitter Id
E-MTAB-8627:Sample 25
age
67
broker name
ArrayExpress
cell type
hepatocyte
common name
human
developmental stage
adult
individual
HF226
organism part
liver
replicate
1
sample name
E-MTAB-8627:Sample 25
scientific_name
Homo sapiens
sex
male