GSM7804571: MDF from KI-WT mice treated with Nutlin - Replicate  3; Mus musculus; OTHER
500,000 MDFs were plated and left to settle for 24 h in an incubator with 10% CO2 at 37°C. Medium was replaced with medium containing nutlin-3a (10 μM, Cayman Chemicals #18585) or DMSO (vehicle control) and cells incubated for 6 h. Adherent cells were dissociated with trypsin (0.25 mg/mL, Sigma #T4174), keeping both the supernatant and the trypsinised fractions for analysis. CUT&RUN was performed using the CUTANA ChIC/CUT&RUN kit v2 (Epicypher, 14-1408) as per the manufacture's protocol. Nuceli were isolated prior to the start of the assay. Nuclei were bound to activated conA beads. Samples were incubated with 0.5 μg of anti-FLAG antibody (Sigma, F1804) overnight. DNA was cut using pAG-MNase, and unbound fragments washed away. Bound chromatin was release from the beads, and DNA was purified. Total recovered DNA was taken into library preparation using the NEBNext Ultra II DNA library Prep Kit for Illumina (#E7645L). CUT&RUN