Stempro adipogenesis media

Cells were harvested following 24-hr exposure to DMSO or 10uM of CMP12 (generous gift of Jun Qi) followed by 24-hr exposure to DMSO plus base media or StemPro Adipogenesis media (ThermoFisher) or 10uM CMP12 plus base media or StemPro Adipogenesis media. CUT&RUN experiments were performed according to established protocols (Skene et al., 2018 (link)) with minor modifications. Briefly, 500K cells per sample were incubated with Concanavalin A-coated beads (Polysciences) for 10 min and then permeabilized with Triton X-100. Cell-bead complexes were then incubated with the indicated antibody for 2 hr at 4°C. Cell-bead complexes were washed and then incubated with pA-MNase for 1 hr at 4°C. Cell-bead complexes were washed and then cooled to ~0°C. pA-MNase was activated by the addition of calcium chloride and incubated for 30 min at ~0°C. The cleavage reaction was quenched with a solution containing spike-in DNA and RNAase A. Cleaved DNA fragments released into solution were then collected and treated with sodium dodecyl sulfate (SDS) and proteinase K (Thermo Scientific). DNA was then isolated using phenol-chloroform. CUT&RUN libraries were prepared with Illumina’s NEBNext Ultra II DNA library Prep Kit with modifications that aim to preserve short fragments.