2024-12-09
ENA first public
ERC000011
ENA-CHECKLIST
CRUK Cambridge Institute, University of Cambridge
INSDC center name
public
INSDC status
ERS19897161
SRA accession
E-MTAB-14079:ko_brd4_b1_t1
Submitter Id
ArrayExpress
broker name
U-2 OS
cell_line
epithelial
cell_type
not collected
collection date
human
common name
Protocols: Bulk CUT&Tag was performed as described previously (Kaya-Okur et al., 2020), with some modifications. Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific, 65601) were incubated with Concanavalin A (biotin conjugate, Sigma-Aldrich C2272) in ratio 10 mg/mL beads to 1.15 mg/mL Concanavalin A at 22 ºC for 30 min at 400 rpm on a thermoshaker (Eppendorf). Volume sufficient for 10µl beads per reaction was prepared. Beads were washed with 1ml of binding buffer (20mM HEPES pH 7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2) and resuspended in a volume of binding buffer sufficient for 10µl beads per reaction. U2-OS cells were harvested with StemPro Accutase (Thermo Fisher Scientific, A1110501) and 2x106 cells were fixed in 0.1% formaldehyde (Thermo Scientific, 28906) in 1ml 1X PBS for 2 min at room temperature and quenched with 0.075M glycine. Cells were centrifuged at 1300g 4 ºC for 4 min and resuspended in wash buffer (20mM HEPES pH 7.5, 150mM KCl, 0.5mM spermidine (Sigma, S0266), one tablet protease inhibitor (Roche cOmplete, EDTA-free, 11873580001)) at 500 cells/ µl. 10µl beads were incubated with 100µl of cell suspension at 25 ºC for 10 min at 600 rpm, washed twice with 100µl wash buffer and resuspended in 50µl of antibody buffer (2mM EDTA, 0.1% BSA (Sigma A8577), 0.05% digitonin (Millipore, 300410) in wash buffer). All wash steps were performed on a DynaMag 2 magnet (Invitrogen). Primary antibody against KLF5, BRD4  and Normal Rabbit IgG were added to each sample at final concentration of (0.2 mg/mL) and incubated at 4 ºC overnight at 600 rpm. Cells were washed twice with 100µl dig-wash buffer (0.05% digitonin in wash buffer) and resuspended in 50µl of dig-wash buffer. 0.5µl of secondary antibody Guinea Pig anti-rabbit IgG (Antibodies-online, ABIN101961) was then added to each sample and incubated at 25 ºC for 1h at 600 rpm. Cells were then washed three times with 500µl dig-wash buffer and resuspended in 50µl dig-300 buffer (20mM 20mM HEPES pH 7.5, 300mM KCl, 0.5mM spermidine, 0.01% digitonin, one tablet protease inhibitor). Mosaic-end adaptor loaded pA-Tn5 (made in house, as described previously; (Kaya-Okur et al., 2020) was then added to each sample in final 1:250 dilution to each sample and incubated at 25 ºC for 1h at 600 rpm. Cells were washed three times with 500µl dig-300 buffer and resuspended in 300µl tagmentation buffer (10mM MgCl2 in dig-300 buffer) and incubated at 37 ºC 1h at 600 rpm.  Cells were then washed twice with 500µl TAPS wash buffer (10mM TAPS (Thermo Fisher Scientific J63268.AE) and resuspended in 100 µL of proteinase K solution (0.5 mg/mL Proteinase K (Thermo Fisher Scientific, EO0491), 0.5% SDS in 10mM Tris-HCl pH 8.0). Samples were vortexed on full and incubated at 55 ºC for 1 hour at 800 rpm. DNA was extracted with DNA Clean & Concentrator 5 (Zymo Research, D4013) following the manufacturer’s instructions, and DNA was eluted in 25µl DNA elution buffer.  Sequencing libraries were generated by mixing 25µl NEBNext High Fidelity 2X PCR Master Mix (New England Biolabs, M0541), 2µl of P5 indexing primer (10 µM), 2µl of P7 indexing primer (10 µM) with 21µl of purified DNA in a PCR reaction under the following conditions: 72 ºC for 5 min, 98 ºC for 30 sec followed by 10 cycles of 98 ºC 10 sec, 63 ºC for 10 sec, and a final extension at 72 ºC for 1 min. Libraries were purified with Ampure XP beads (Beckman Coulter, A63882) by incubation with 0.4X ratio of beads for 15 mins, followed by supernatant incubation with 1.4X ratio of beads for 15 mins. Beads were washed twice with 800µl 80% ethanol and eluted in 25µl of 10mM Tris-HCl pH 8.0. Library size distribution was analysed on a Tapestation 2200 (Agilent Technologies), and library concentrations were quantified by NEBNext Library Quant Kit for Illumina (New England BioLabs, E7630S).
description
young adult
developmental stage
osteosarcoma
disease
KLF5-/-
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
bone
organism part
Homo sapiens
scientific_name
ko_brd4_b1_t1
title