NextSeq 550 sequencing; mRNA-seq based gene expression profiling of differentiating murine brown preadipocytes (IBA)
mRNA-seq based gene expression profiling of differentiating murine brown preadipocytes (IBA)
Murine derived brown pre-adipocytes (IBA) were cultured in high-glucose Dulbeccos modified Eagles medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS, AMIMED), with 100x penicillin/streptomycin/glutamine (100x Pen/Strep/Glutamine, Life Technologies) in a 5% CO2 humidified atmosphere at 37C and maintained at less than 80% confluence before passaging Differentiation of IBAs was induced by exposing confluent cells (Day 0) to DMEM containing 10% FCS supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 uM dexamethasone, 1.96 nM insulin (Sigma, stock solution of 167uM prepared in PBS), 125 uM indomethacin (Sigma, stock solution of 18mM prepared in absolute ethanol), 1nM T3 (3,3,5-Triiodo-L-thyronine sodium salt, Sigma, prepared in 45mM KOH). After exactly 28 hours, the induction medium was replaced with adipocyte differentiation medium consisting of 1nM T3 and 1.96 nM insulin Total RNA was extracted using a Qiagen RNAeasy plus mini kit/ Zymo Direct-zol kit according to manufacturers protocol by using either the RLT buffer lysis system or the TRIzol/Choloroform extraction procedure (Sigma) respectively. The RNA concentration and quality was determined using a nanodrop (1.8  A260/A280  2.2) and by visual inspection of separated bands on Fragment Analyzer (Advanced Analytical).  Illumina Truseq RNA sample preparation kit v2 protocol (Illumina, USA) was followed using 500 ng of RNA per sample as starting material. Libraries were checked for quality and quantified using the Bioanalyzer 2100 (Agilent, USA), before being sequenced (single end sequencing at UNIL; and GECF, EPFL). Two libraries (technical replicates 1 and 2) were prepared according to the un-stranded Illumina Truseq RNA sample preparation kit v2 protocol while the second biological replicate was prepared using the stranded protocol.
Experimental Factor: developmental stage
maturing brown adipocyte
Experimental Factor: time
2