title: 2_CTCF_WntOFF_dBcat
organism: Homo sapiens
description: Protocols: CUT&RUN for CTCF was performed as described in (PMID: 29651053), with minor modifications as described here. Three independent rounds of experiments were performed for the three biological replicates of each cell line and condition. 250,000 cells were used per replicate. Antibodies used included anti-CTCF (abcam, ab70303) and anti-HA (Merck, 05-902R) at 1:100 dilutions. For each replicate of CTCF, a corresponding anti-HA negative control was performed from cells in the same well. 40 µl of ConA beads were used per sample, and digitonin concentration was 0.025% throughout the protocol. CUT&RUN LoV-U for H3K4me3, H3K4me1 and H3K27ac was performed according to (PMID: 36355069), based originally on (PMID: 29651053), with minor modifications as described here. Experiments were performed in duplicate for each condition. 250,000 cells were used per sample and bound to 10 µl magnetic ConA agarose beads. Antibodies used included anti-H3K4me3 (antibodies online, ABIN6971977), anti-H3K4me1 (antibodies online, ABIN3023251), and anti-H3K27ac (antibodies online, ABIN2668475) at 1:100 dilutions. The digestion buffer was kept and added to the release fraction before DNA purification. 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. DNA was purified with phenol:chloroform:isoamyl alcohol followed by ethanol precipitation, and stored until library preparation. Library preparation was performed according to PMID: 36355069 using the KAPA Hyperprep Kit (KAPA Biosystems, Cat. #KK8504) with the following modifications: for CTCF samples, 15 cycles of amplification were performed, while LoV-U samples received 13 cycles as previously described. For CTCF samples, size selection was performed post-amplification using 2% E-Gel Size Select II Gels (Thermo Fisher, Cat #G661012), retrieving fragments from 150 – 240 bp (representing 30 – 120 bp without the adapter sequences).
attributes.tag: disease
attributes.value: normal
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: collection date
attributes.value: not collected
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: kidney
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: genotype
attributes.value: WntOFF_dBcat
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2024-02-16T00:32:08Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2024-02-16T00:32:08Z
attributes.tag: cell_line
attributes.value: HEK293T
attributes.tag: cell_type
attributes.value: kidney cell
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2024-02-16
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2024-02-16