title: ChIP-seq of Irx3 in primary cells from mouse gWAT and iWAT on days -1 and 1 of adipogenic differentiation
library_construction_protocol: Mouse inguinal and gonadal white adipose tissues where excised from 6-10 weeks old C57BL/6NJ (B6N) mice fed ad libitum with SDS maintenance chow (RM3, 3.6 kcal/g).  Primary preadipocytes were isolated from the WAT depots by by collagenase digestion and centrifugation to separate floating adipocytes from the SVF pellet. The SVF was resuspended in DMEM GlutaMax culture medium supplemented with 10% FBS and 1% PEST. The cell suspension was filtered through a 40 um mesh and pooled cells from 6-12 mice were seeded on a 10 cm dish at a density of 100.000 cells/mL.  Cells were grown to confluence, and induced to differentiate 2 days post confluency by addition of 0.5 mM IBMX, 1 uM Dex, 5 ug/mL human Insulin to the growth media.  On days -1 and 1 of differentiation, cells were fixated by addition of 1/10 volume of freshly prepared formaldehyde solution (11% formaldehyde, 0.1 M NaCl, 1mM EDTA pH 8.0 and 50 mM HEPES pH 7.9) to the culture media. After 15 minutes, the fixation reaction was stopped by addition of 1/20 volume of 2.5 M glycine. The cells were thereafter kept on ice and washed twice with 0.5% Igepal in PBS. In the second wash, 1mM PMSF was added. Pellets were snap-frozen in liquid nitrogen and stored at -80oC until shipment on dry ice to Active Motif for ChIP-sequencing   Nucleic acid extraction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52. Nucleic acid library construction was performed by Active Motif by in-house protocols based on public protocols:Park, P.J., 2009. ChIP–seq: advantages and challenges of a maturing technology. Nature Reviews Genetics 10: 669–80.Pepke, S., Wold, B., Mortazavi, A., 2009. Computation for chip-seq and rna-seq studies. Nature Methods 6(11 Suppl): S22-32.Furey, T.S., 2012. ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nature Reviews Genetics 13(12): 840–52.