title: Illumina HiSeq 2500 sequencing; RNA-seq of coding RNA (polysomal and free fraction) from human adipose tissue stem cells treated with cyclopamine, purmorphamine and DMSO
design: RNA-seq of coding RNA (polysomal and free fraction) from human adipose tissue stem cells treated with cyclopamine, purmorphamine and DMSO
library_construction_protocol: Polysomal and Free fractions from adipose tissue stem cells cultures treated with cyclopamine, purmorphamine or DMSO for 24 hours were prepared according to (Spangenberg et al., 2013). In brief, cells were treated with 0.1 mg/mL cycloheximide (Sigma-Aldrich) for 10 min at 37oC, removed from the culture flasks with a cell scraper, and resuspended in 0.1 mg/mL cycloheximide in PBS. The suspension was centrifuged (2,000 g for 5 min), and the resulting pellet was washed twice with 0.1 mg/mL cycloheximide in PBS. The cells were lysed by incubation for 10 min on ice with polysome buffer (15mM Tris-HCl pH 7.4, 1% Triton X-100, 15mM MgCl2, 0.3M NaCl, 0.1 mg/mL cycloheximide), and the cell lysate was centrifuged at 12,000 g for 10 min at 4oC. The supernatant was carefully isolated, loaded onto 10% to 50% sucrose gradients, and centrifuged at 39,000 rpm (HIMAC CP80WX HITACHI) for 160 min at 4oC. The sucrose gradient was fractionated with the ISCO gradient fractionation system (ISCO Model 160 gradient former), connected to a UV detector to monitor the absorbance at 275 nm, and the polysome profile was recorded. The free and polysomal RNA fractions were extracted by DirectZOL (Zymo Research).  Cells and culture conditions Human Adipose-Derived Stem Cells (ASC) from lipoaspirates were purchased from a commercial source (Lonza®, Walkersville, USA; catalog number PT-5006). ADSC from 3 different donnors were cultured in accordance with datasheet instructions. In brief, cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco Invitrogen®, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco Invitrogen®, Carlsbad, California, USA) and 4 mM L-glutamine (Gibco Invitrogen®, Carlsbad, California, USA), without antibiotics. The cells were maintained at 37 C in 5% CO2 atmosphere.  Cyclopamine (Sigma Aldrich - C4116) was dissolved in dimethyl sulfoxide, stocked 1mM and used at 5µM in culture for 24 hours. Purmorphamine (Sigma Aldrich – SML0868) was dissolved in dimethyl sulfoxide, stocked 1mM and used at 1µM in culture for 24 hours. The RNA from polysome and free fractions was extracted using DirectZol kit (Zymo).  Illumina® TruSeq® Stranded Total RNA Sample Preparation kits
attributes.tag: Experimental Factor: compound
attributes.value: dimethyl sulfoxide
attributes.tag: Experimental Factor: fraction
attributes.value: polysome associated RNA