2025-01-28
ENA first public
ERC000011
ENA-CHECKLIST
Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden
INSDC center name
public
INSDC status
ERS22987810
SRA accession
E-MTAB-13727_2:4_RAD21_WntON_WT
Submitter Id
ArrayExpress
broker name
HEK293T
cell_line
kidney cell
cell_type
not collected
collection date
human
common name
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.  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).
description
embryo stage
developmental stage
none
disease
WT
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
kidney
organism part
Homo sapiens
scientific_name
4_RAD21_WntON_WT
title