Illumina HiSeq 1500 sequencing; Transcriptional profiling of hepatic stellate cells (HSCs) isolated from Western diet/high fructose-fed C57BL6/J mice, carbon tretrachloride (CCl4)-treated C57BL6/J mice, and of murine HSCs differentiated in vitro
Transcriptional profiling of hepatic stellate cells (HSCs) isolated from Western diet/high fructose-fed C57BL6/J mice, carbon tretrachloride (CCl4)-treated C57BL6/J mice, and of murine HSCs differentiated in vitro
FACS-sorted HSCs were cultivated for 2 hrs (control), 12 hrs, 1, 4, 8 or 12 days in technical triplicates in Falcon 12-well Polystyrene culture plates (50000 cells/well). HSCs were kept in Dulbecco's Modified Eagle's Medium (DMEM; #52-100-021, GIBCO) supplemented with 10% calf serum (B15-005, PAA), 100U Penicillin, 100 µg/mL Streptomycin (#DE 17-602E, Lonza), and 1.25 µg/mL Amphotericin B (15290-018, Gibco). Control cells were kept in suspension in culture medium for 2 hrs before centrifugation and snap freezing. Medium was replaced every two days. In vitro treatments (U0126):  For MEK inhibition, U0126 (10 µM in DMSO; #9903, Cell Signaling Technology) or DMSO (0.05%) was added to the medium from day 2 until harvest on day 6 post isolation. Fresh medium with U0126 or DMSO was added every two days. Whole mouse liver was perfused with collagenase and protease. Single cell suspension was stained in 70micron cell strainer. Hepatic stellate cells were enriched by density centrifugation and FACS sorting of retinoid-positive, Sytox-red negative (live) cells. HSCs were lyzed in RLT lysis buffer from RNeasy Mini Kit (Qiagen) and total RNA extracted according to manufacturer's instructions after a standard phenol/chloroform extraction. RNA concentrations were measured using a Qubit 3.0 Fluorometer (Invitrogen) and quality of the RNA was assessed using a Bioanalyzer 2100 (Agilent Technologies). Purified RNA (3-4 biological replicates) was polyA-selected and subjected to fragmentation before cDNA synthesis. For library construction, we used the NEBNext Ultra™ RNA Library Prep Kit for Illumina. and sequenced using the Illumina HiSeq 1500 platform.
Experimental Factor: protocol
in vitro; differentiation
Experimental Factor: diet
not applicable
Experimental Factor: compound
none
Experimental Factor: time
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