2_HiChIP_CTCF_WntON_WT
Homo sapiens
Protocols: Flasks were taken from the incubator and fresh methanol-free formaldehyde 16% was added to the culture media to a final concentration of 1%. Fixation proceed at RT for 15 minutes, and was then quenched with cold glycine to 150 mM and incubated for 5 min RT. Media was removed and cells were washed twice with cold PBS. Cells were harvested in PBS using a scraper, transferred to a chilled tube, and pelleted at 4 dgrees for 5 minutes. Supernatant was discarded and the cell pellet was flash frozen and store at -80 degrees until HiChIP. HEK293T human embryonic kidney cells (parental) and Δβ-catenin cells (generated in PMID: 30425074) were cultured in a 37 °C incubator in 5% CO2 and 89% humidity. Culture medium used was high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) supplemented with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco). Cells were stimulated either with 10 µM CHIR99021 (Wnt ON) or 1 nM LGK (Wnt OFF) for 24 hr prior to harvest for assays. HiChIP was performed in duplicates in both LGK (Wnt-off) and CHIR99021 (Wnt ON) cell lines using the Arima-HiC+ Kit (A101020) and according to the guidelines in the Arima-HiChIP user guide for mammalian cells. HEK293T cells were cultured as described above and stimulated for 24 hours for Wnt ON and Wnt OFF, and approximately 5 × 106 cells per line were used to obtain at least 15 μg of input DNA for HiChIP. Cells were fixed in 1% fresh methanol-free formaldehyde for 15 min at room temperature while rotating, and Glycine was used to quench the crosslinking reaction (final concentration 125 mM Glycine) for 5 min. After two washes with cold PBS, crosslinked cells were harvested in PBS by scraping, pelleted and stored at -80°C until further used. In the subsequent steps, crosslinked chromatin was digested with restriction enzymes, end-filled with biotinylated nucleotides and ligated. Proximally ligated chromatin was then sheared on a Covaris E22O instrument, with the following shearing parameters: shearing time 5 min, PIP 105, duty factor 5, and 200 cycles per burst), to achieve a fragment size range of 200 bp to 800 bp. Chromatin immunoprecipitation was performed with an antibody against CTCF (AbFlex CTCF , Active Motif, Catalog Nos:91285, Lot No:31321002), using 0.5 g of antibody per g of sheared chromatin. After biotin enrichment and adapters ligation, immunoprecipitated DNA was subjected to PCR amplification (18 cycles) using Accel-NGS 2 S Plus DNA Library Kit (#21024, Swift Biosciences) and indexing Kit (#26696, Swift Biosciences), according to the Arima-HiChIP Library Prep user guide.
disease
normal
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
kidney
developmental stage
embryo stage
genotype
WntON_WT
isolate
not applicable
ENA-FIRST-PUBLIC
2024-02-16T00:32:09Z
ENA-LAST-UPDATE
2024-02-16T00:32:09Z
cell_line
HEK293T
cell_type
kidney cell
geographic location (country and/or sea)
not collected
ENA-FIRST-PUBLIC
2024-02-16
ENA-LAST-UPDATE
2024-02-16