Illumina HiSeq 4000 paired end sequencing; Time-course RNA-Seq of 3T3-L1 cells as they undergo adipogenesis, with both control samples and samples treated with Isoproterenol
Time-course RNA-Seq of 3T3-L1 cells as they undergo adipogenesis, with both control samples and samples treated with Isoproterenol
Cells were washed in PBS, lysed by addition of RLT buffer (Qiagen, Hilden, Germany) and homogenized by centrifugation on QIAShredder columns (Qiagen) before snap-freezing in liquid nitrogen.  Mouse embryonic fibroblast Rb-/- Line 3 (ME3) cells were previously generated (41) and characterized to be a model of beige adipocytes (42–46). Cells were  grown in AmnioMAX -C100 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 7.5% FBS (Thermo Fisher Scientific, Waltham, MA, USA ), 7.5% C100 (Thermo Fisher Scientific), 1% penicillin-streptomycin (PEST) (Sigma, St. Louis, MO, USA) and 2 mM L-glutamine (Sigma) at 37oC and 5% CO2. Cells were initiated to differentiate three days post confluency (day 0) by induction medium containing 5 ug/mL Insulin (INS) (Sigma), 1 μM Dexametasone (DEX) (Sigma), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma) and 1 μM Rosiglitazone (ROSI) (Cayman Chemical, ann Arbor, MI, USA). From day 2 to day 4 only insulin was added to the basal medium and from day 4 to 7 cells were grown in the basal medium. RNA was isolated using the RNeasy Mini kit (Qiagen) with on-column DNaseI-treatment and RNA integrity was validated by the RNA 6000 Nano kit on the 2100 BioAnalyzer (Agilent, Santa Clara, USA). Library preparation and 2x75bp paired-end mRNA sequencing was performed using the TruSeq Stranded mRNA kit (Illumina, Sand Diego, CA, USA)
Experimental Factor: time
7
Experimental Factor: phenotype
differentiated
Experimental Factor: stimulus
water