title: GSM8285194: input, Rosi, biological replicate1; Mus musculus; OTHER
library_construction_protocol: Stromal vascular fractions were isolated as previously described. Briefly, entire WAT depots were cut into small pieces, suspended in DMEM (GIBCO) + 50 mM HEPES (GIBCO) + 1mg/mL type II collagenase (Sigma), + 1% BSA - low fatty acid (Gemini), and digested for 30 minutes at 37°C with agitation (120 rpm). The resulting dissociated tissue was passed through 100μm strainers, and adipocytes were removed from the supernatant by centrifugation (750g, 10min, 4C). After a second filtration with 40μm strainers, red blood cells were lysed using ACK lysis buffer (GIBCO). Cells were then treated to differentiate with complete adipogenic cocktail (0.5mM 3-isobutyl-1-methylxanthine (Sigma), 1μM dexamethasone (Sigma), 5μg/mL insulin (GIBCO) plus either 1μM rosiglitazone (Novo Nordisk) or vehicle (DMSO) for 2 days. After that, media was replaced for minimal maintenance media (Insulin/Rosi or Insulin/DMSO) for 2 days. At day 4 of differentiation cells were boosted with fresh media for one hour and for the last 5 minutes cycloheximide (Sigma) was added to the media (final concentration 100μg/mL). Cells were lysed and collected in hypotonic lysis buffer (5mM Tris HCl pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 100 μg/mL cycloheximide, 2 mM DTT), and 0.5% Triton, 0.5% Sodium Deoxycholate were then added to the cell lysates after collection. The cytoplasmic extracts were resolved on 5-50% sucrose gradients by centrifugation in an SW40 rotor at 150,000 x g for 2 hours. The absorbance at 260 nm was measured using a Piston Gradient Fractionator Model 153 instrument (BioComp, Canada). RNA from polysomes or total RNA pool was resuspended in Trizol reagent (ThermoFisher), and RNA was isolated using Direct-zol RNA Microprep kits as described by the manufacture (Zymo research). The RNA sample received was quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using TapeStation (Agilent Technologies, Palo Alto, CA, USA). The RNA sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina using manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First-strand and second-strand cDNA were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing library was clustered on three lanes of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer's instructions. The sample was sequenced using a 2x150bp Paired End (PE) configuration.