Q100-02
Mus musculus
Protocols: On day of harvest, cells were collection by trypsination, centrifugation and snap-freezing in liquid N2 on days -1, 1 and 9, respectively. Cell pellets were kept at -80C until RNA isolation. ME3 control and Irx3-KO mouse beige-like preadipocytes were grown in proliferation medium (Amniomax -C100, 1% PEST, 7.5% FBS, 7.5% C100 and 1% L-glut) in 6-well plates pre-coated with 0.1% gelatine until confluence (day -2), before induction of adipogenesis on day 0 followed by adipogenic differentiation for 9 days.   Cells were treated with Induction medium (861 nM Insulin, 1 uM Dexamethasone, 0.5 mM IBMX and 1 uM Rosiglitazone added to the prolif medium) from day 0-2, followed by differentiation medium (only 861 nM Insulin added to the prolif medium) from day 2-4 and proliferation medium from day 4-9. Cells were treated with either DMSO or 0.5 uM ML-792 for 24h either from day -2 to day -1 or day 0 to day 1, or throughout the differentiation from day 0 to 9 (n =3 replicate wells for each condition) before collection by trypsination, centrifugation and snap-freezing in liquid N2 on days -1, 1 and 9, respectively. Cell pellets were kept at -80C until RNA isolation. RNA was isolated using phenol/guanidine-based lysis followed by chloroform extraction and silica-based spin column purification of the aquous phase according to instructions of the RNeasy Lipid Tissue kit (Qiagen). RNA quantity and integrity was assessed by Qubit fluorometric quantification.    Library prep: Illumina Stranded mRNA, Ligation kit  Quantification of libraries: Illumina MiSeq Nano sequencing Library QC on Agilent TapeStation