ZSCAN4
Protocols: 30 million 293T 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). 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher, 41966–029) supplemented with 10% foetal calf serum (Merck, F9665-500ML, Lot 19A124) and 1X Penicillin-Streptomycin-L-Glutamine (Corning, 30-009-CI). Cells were cultured in 1 µg/ml doxycycline-containing culture medium for 72 h prior to harvest to induce overexpression of a transgene under a tetracycline-response element. 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 then immunoprecipitated with antibody-bound Dynabeads Protein G (Thermo Fisher, 10004D) overnight, washed with high-salt wash buffer, low-salt wash buffer, and LiCl wash buffer. The chromatin 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 10 ng of it was used for library preparation with NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs, E7546L).
isolate
not applicable
organism
Homo sapiens
organism
Homo sapiens
cell_line
293T
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
kidney
cell_type
epithelial cell
geographic location (country and/or sea)
not collected