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
WD experiments: mice were fed WD (829100, SDS Research Diet) supplemented with fructose (42g/L) in drinking water for 6, 12, 16 or 24 weeks. Control mice received standard chow diet (altromin #1324) and regular drinking water. WD feeding was initiated so that all mice were 30 weeks of age at the end of the experiment. CCl4 treatment: After over-night (o.n.) fasting, mice were given CCl4 (1mL/kg; 289116, Sigma-Aldrich) diluted in corn oil at a 1:4 ratio twice weekly by oral gavage for 1, 4 or 8 weeks. Control mice received same amount of corn oil as the CCl4-treated mice and were fasted accordingly. 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 vivo; western diet
Experimental Factor: diet
western diet/fructose
Experimental Factor: compound
none
Experimental Factor: time
16