FKBP12(F36V)-ZNF24_clone1_dTAGv1
Protocols: 1.5 million HAP1 cells were detached by incubating in trypsin for 2 min. 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 or an indicated length of time prior to harvest. Nuclear extraction and permeabilisation were performed on the detached cells in ATAC-RSB buffer containing 0.1% NP-40 and 0.1% Tween-20 on ice for 3 min. The nuclei were washed once with the same buffer and digested with 1 U MNase (Worthington Biochemical Corp., LS004798) in 50 µl buffer containing 1 mM CaCl2 at 37°C for 20 min on a thermomixer with 1,000 rpm mixing. The digested DNA was cleaned-up with DNA Clean and Concentrator-5 Kit (Zymo Research, D4014). The purified DNA was size-selected in a 1.5% agarose gel for fragments smaller than 200 bp. The DNA was then purified from gel, and 500 ng of it was used as input for library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, E7645S).
isolate
not applicable
organism
Homo sapiens
organism
Homo sapiens
cell_line
HAP-1
collection date
not collected
scientific_name
Homo sapiens
common name
human
geographic location (country and/or sea)
not collected