5 protocols
nucleic acid sequencing protocol
SOLiD Full-Scale Template Bead preparation protocol
nucleic acid library construction protocol
We subjected total and polysome-associated RNA samples to amplification with the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion), to provide a template for SOLiD libraries. The cDNA libraries were prepared with the SOLiD Whole Transcriptome Analysis Kit and the purified products were evaluated with an Agilent Bioanalyzer (Agilent). Library molecules were subjected to clonal amplification according to the SOLiD Full-Scale Template Bead preparation protocol and sequenced with the SOLiD4 System (Applied Biosystems).
nucleic acid extraction protocol
Polysomal fractions were prepared with a modified version of the procedure described by Holetz et al. In brief, hASC cultures at 50 to 60% confluence were treated with 0.1 mg/ml cycloheximide (Sigma-Aldrich) for 10 minutes at 37¡C. The cells were removed from the culture flasks with a cell scraper and resuspended in 0.1 mg/ml cycloheximide in PBS. The suspension was centrifuged (2000 x g for 5 minutes) and the resulting pellet was washed twice with 0.1 mg/ml cycloheximide in PBS. The cells were lysed by incubation for 10 minutes on ice with polysome buffer (15 mM Tris-HCl pH 7.4, 1% Triton X-100, 15 mM MgCl2, 0.3 M NaCl, 0.1 _g/ml cycloheximide, 1 mg/ml heparin). The cell lysate was centrifuged at 12000 x g for 10 minutes at 4¡C. The supernatant was carefully isolated, loaded onto 10% to 50% sucrose gradients and centrifuged at 39000 rpm (SW40 rotor, HIMAC CP80WX HITACHI) for 160 minutes at 4¡C. The sucrose gradient was fractionated with the ISCO gradient fractionation system (ISCO Model 160 gradient former), connected to a UV detector for the monitoring of absorbance at 275 nm, and the polysome profile was recorded.
nucleic acid extraction protocol
The total and polysomal RNA fractions were extracted by a standard Trizol (Invitrogen) RNA isolation protocol.
growth protocol
Stem cells were obtained from adipose tissue from obese human donors ( two males and one female, ages: 41, 52, 23) . All samples were isolated, collected after informed consent had been obtained. hASCs were isolated, cultured and characterized as following. Briefly, 100 ml of adipose tissue was washed with sterile phosphate-buffered saline (PBS) (Gibco Invitrogen). A one-step digestion by 1 mg/ml collagenase type I (Invitrogen) was performed for 30 mins at 37 degrees C during permanent shaking and was followed by filtration through first a 100- and then 40-um mesh filter (BD FALCON, BD Biosciences Discovery Labware, Bedford, MA, USA).