male gWAT mature
Mus musculus
Protocols: 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, 0, 0,4h, 1, 2, 7 and mature stage, cells lysed directly in the cell culture plate and nuclei released. Lysis buffer was gently pipetted up and down to wash nuclei off the well and transferred into a chilled 1.5ml tube to create crude nuclei. Nuclei were spun down at 600 x g for 10 min at 4℃, nuclei pellets were then re-suspended in 40 µl Tagmentation DNA (TD) Buffer. Transposition reaction was performed as previously described (Buenrostro et al. 2015). All tagmented DNA was PCR amplified for 11-13 cycles. Quality was assessed using a DNA1000 Chip (Applied Biosystems) and run on a Bioanalyzer (Applied Biosystems). The profiles showed that all libraries had a mean fragment size of ~190 bp and characteristic nucleosome patterning, indicating good quality of the libraries.