title: HCT116_B
organism: Homo sapiens
description: Protocols: Cells were filtered through a 40 µm cell strainer (KKE3.1, Carl Roth) and processed for CUT&RUN (Zambanini et al., 2022). 500,000 cells/sample were harvested using either Trypsin EDTA 0.25% (HEK293Ts) or EDTA 0.5 mM in PBS (hESCs) and washed twice in DPBS (Cat. #14190094, Thermo Fisher Scientific). HEK293T cells (HEK293Ts) were cultured in medium consisting of DMEM, high glucose (41966-029, Gibco - Thermo Fisher Scientific) supplemented with 10% Calf Bovine Serum (12133C, Sigma-Aldrich) and 10 U/ml Penicillin-Streptomycin (15276355, Gibco - Thermo Fisher Scientific). HEK293Ts were passaged by incubation in Trypsin EDTA 0.25% (25-200-056, Thermo Fisher Scientific). hESCs were cultured in Essential 8TM medium (A1517001, ThermoFisher Scientific) in plates and flasks coated with 10ug/ml Vitronectin (A14700, ThermoFisher Scientific). Cells were passaged by incubation in 0.5 mM EDTA-PBS (stock: 0.5 M EDTA, AM9260G, Thermo Fisher Scientific). HEK293Ts were grown in culture medium supplemented with 10 nM LGK (S7143, Selleck Chemicals) for the 48 hours preceding the seeding. HEK293Ts were then seeded at a density of 104 cells/cm2 in culture medium (see “Cell Culture” section) supplemented with 10 nM LGK. After 24 hours, cells were cultured in medium supplemented with 10 mM CHIR99021 (SML1046, Sigma Aldrich). Medium and treatment were renewed every 24 hours. hESCs were seeded at a density of 104 cells/cm2 in Essential 8TM medium, and after 24 hours, they were cultured in Essential 8TM medium supplemented with 10 uM CHIR99021. Cells were then detached (as described in the “Sample collection protocol”) after 90 minutes, 4 hours, 24 hours, and 72 hours of CHIR treatment. All experiments were performed in triplicate (n = 3 for each cell type at each time-point). Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermi- dine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), then resuspended in 40 μl NE per sample and bound to 20 μl Magnetic ConA Agarose beads equilibrated in binding buffer. After incubation, nuclei and beads were resuspended for 5 minutes in EDTA wash buffer (wash buffer with EDTA [0.2 mM]). Samples were divided in 200 μl PCR tubes and antibody incubation was performed in 200 μl wash buffer with 2 μl of anti-β-catenin antibody (Cat. #ABIN2855042, antibodies-online) overnight (ON) at 4 °C on a rotator. After ON incubation, samples were washed five times in wash buffer and resuspended in 200 μl of pAG-MN buffer (wash buffer supplemented with pAG-MN 0.6 μg/ml) for 30 minutes at 4 °C on a rotator. Samples were washed five times, followed by digestion for 30 minutes in wet ice in wash buffer with 2 mM CaCl2. After 30 minutes, the digestion buffer was removed and the reaction was stopped with 50 μl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and the samples were incubated for 1 hour at 4°C. Beads were collected on the magnetic rack, while the liquid was transferred to a new PCR tube, where it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio- Tek) at 2X, and then resuspended in 20 μl Tris-HCl pH 7.5. Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat. #KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications. End repair and A-tailing was performed with 0.4 size reactions with 20 µl of purified DNA. The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was done with 0.4 size reactions. KAPA Dual Indexed adapters were used at 0.15 µM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2X. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed with 0.5 size reactions. The cycling conditions were set as follows: 45 sec initial denaturation at 98 °C, 15 sec denaturation at 98 °C, 10 sec annealing/elongation at 60 °C, 1 min final extension at 72 °C, hold at 4 °C, with 13 cycles. After amplification, a post-amplification cleanup was performed with 1.2X beads. Libraries were then run on an E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were cut out and purified using the GeneJET Gel Extraction Kit (Cat. #K0691, Thermo Scientific) according to manufacturer's instructions.
attributes.tag: disease
attributes.value: colorectal cancer
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-22
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-22
attributes.tag: cell_line
attributes.value: HCT116
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: colon
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-22
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-22