title: RNA-seq of mouse 3T3-L1 cell line treated with CX-4945 compared to untreated controls during differentiation
design: RNA-seq of mouse 3T3-L1 cell line treated with CX-4945 compared to untreated controls during differentiation
library_construction_protocol: Cells were washed in PBS and harvested for RNA extraction 7 days after adipogenesis induction. Harvesting was done in duplicates in 3 separate experiments. Cells were cultured at 37°C, 5% CO2, in DMEM medium (Gibco, Invitrogen, Taastrup, Denmark) supplemented with fetal bovine serum (10%), and penicillin (100 U/ml)/streptomycin (100 µg/ml). A differentiation cocktail consisting of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, Brøndby, Denmark), 1 µM Dexamethasone (Dex, Sigma-Aldrich), and 5 µg/ml Insulin (Ins, Sigma-Aldrich) was added to confluent cells at day 0. Fresh medium containing 10% FBS and 5 µg/ml Insulin was added to the cells after additional 72 hrs (i.e. day 3). Cells were subsequently supplemented with fresh growth medium on day 5. Where indicated, cells were additionally treated with vehicle (DMSO) or 10 µM CX-4945 (Silmitasertib, Selleck Chemicals, Houston, TX, USA) starting on day 0. Sample preparation was performed as described in the NEBNext ultra II RNA library prep kit (Illumina). The amplified libraries were validated by Agilent 2100 Bioanalyzer using a DNA 1000 kit from Agilent Technologies and quantified by qPCR using the KaPa Library Kits (KaPa Biosystems, Wilmington, MA, USA). Sample preparation was performed as described in the NEBNext ultra II RNA library prep kit (Illumina, San Diego, CA, USA). The amplified libraries were validated by Agilent 2100 Bioanalyzer using a DNA 1000 kit (Agilent Technologies, Inc., Santa Clara, CA, USA) and quantified by qPCR using the KaPa Library Kits (KaPa Biosystems, Wilmington, MA, USA).