FKBP12(F36V)-ZNF24_clone3_dTAGv1_H3K27ac
Protocols: 30 million HAP1 cells were detached with trypsin and were cross-linked with 1% (vol/vol) formaldehyde in PBS for 10 min, and the reaction was quenched with Tris-HCl (200 mM, pH = 8). HAP1 cells (Horizon Discovery, C631) were cultured in IMDM (Thermo Fisher, 12440061) supplemented with 10% foetal calf serum (Merck, F9665-500ML, Lot #19A124) and 1X Penicillin-Streptomycin-L-Glutamine (Corning, 30-009-CI).  To induce the degradation of ZNF24 endogenously tagged with an FKBP12(F36V), the cells were cultured in medium containing either 500 nM dTAGv-1 (MedChemExpress, HY-145514) or the same volume of DMSO for 24 h prior to harvest. The cross-linked nuclei were extracted and sonicated with the Covaris E220 Focused-ultrasonicator with the following settings: 20 min at 5% duty cycle, 140 W, 200 cycles.  The fragmented chromatin was on-bead tagmented with pA-Tn5 (Protein Production and Structure Core Facility, EPFL) on a thermomixer at 37°C for 5 min with 800 rpm shaking. The chromatin was washed twice with low-salt wash buffer and was de-crosslinked with 1 µl Proteinase K (20 mg/ml) at 65°C and 1,400 rpm shaking overnight. DNA was purified with MinElute PCR purification kit (Qiagen, 28006) and was PCR amplified with Q5 High-Fidelity 2X Master Mix (New England Biolabs, M0492L) and indexed primers. The amplified library was cleaned-up by adding 1.2 times the volume of AMPureXP beads (Beckman Coulter, A63882) and eluted with 10 mM Tris-HCl, pH 8.0.
isolate
not applicable
organism
Homo sapiens
organism
Homo sapiens
cell_line
HAP-1
collection date
not collected
scientific_name
Homo sapiens
common name
human
genotype
FKBP12(F36V)-ZNF24
geographic location (country and/or sea)
not collected