FKBP12(F36V)-ZNF24_clone2_dTAGv1
Protocols: 50,000 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, followed by 2.5 µl Tn5 (Illumina, 20034197) tagmentation in a 50 µl reaction at 37°C for 30 min on a thermomixer with 1,000 rpm mixing. The transposed DNA was cleaned-up with DNA Clean and Concentrator-5 Kit (Zymo Research, D4014). The cleaned DNA was PCR amplified with Q5 High-Fidelity 2X Master Mix (New England Biolabs, M0492L) and indexed primers ordered from IDT. The amplified library was double size-selected by 0.5 and 1.5 volumes of AMPureXP beads (Beckman Coulter, A63882) to enrich for fragments ranging from 150-700 bp and was 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
geographic location (country and/or sea)
not collected