W56-31
Mus musculus
Protocols: ME3 cells were differentiated for 0, 1 and 7 days, trypsinated, pelleted, snap-frozen in liquid nitrogen and stored at -80C. Cells were grown in AmnioMAX-C100medium supplementedwith 7.5% FBS, 7.5% C100 (all from Thermo Fisher Scientific,Waltham, MA, USA), 1% penicillin-streptomycin (PEST) (Sigma, St.Louis, MO, USA) and 2 mM L-glutamine (Sigma) at 37 °C and 5% CO2.Adipogenic differentiation was initiated three days post confluency(day 0) by induction medium containing 5 μg/mL Insulin (INS)(Sigma), 1 μMDexamethasone (DEX) (Sigma), 0.5mMisobutylmethylxanthine(IBMX) (Sigma) and 1 μM Rosiglitazone (ROSI) (CaymanChemical, Ann Arbor, MI, USA). From day 2 to day 4 only insulin wasadded to the basal medium and from day 4 to 7 cells were grown inthe basal medium. The cells were thawed on ice, and treated with DNase to remove DNA released from dead cells before lysis in ice-cold ATAC seq lysis buffer from the ATAC-Seq Kit (Active Motif, Carlsbad, CA, USA) for 2 min. Crude nuclei extracts were spun down at 600xg for 10 min at 4C and nuclei were resuspended in 40 µL 1X Tagmentation buffer. The integrity of the nuclei was verified under microscope, counted, and 50,000 nuclei was transferred pr sample for tagmentation. Tagmentation and indexing was performed according to the instructions of the ATAC-seq Kit (Catalog No. 53150, Active Motif). Briefly, nuclei were incubated in tagmentation buffer for 30 min at 37C with gentle mixing every 5 min. Tagmented DNA was purified by a standard silica spin column method provided by the kit. Indexing/PCR amplification of the library performed using unique combinations of i5 and i7 indexing primers. Indexed DNA was cleaned up using SPRI magnetic beads. Library QC was performed by a D5000 ScreenTape assay