2024-12-09
ENA first public
ERC000011
ENA-CHECKLIST
CRUK Cambridge Institute, University of Cambridge
INSDC center name
public
INSDC status
ERS19586380
SRA accession
E-MTAB-14062:KO_UT_2
Submitter Id
ArrayExpress
broker name
U-2 OS
cell_line
epithelial
cell_type
not collected
collection date
human
common name
Protocols: U2-OS cells were seeded in 15-cm plates, and 16h later were either left untreated or treated with ATRi (AZD6736, 1uM, 24h).  Cells were then washed with PBS, lysed in cytoplasmic lysis buffer (50 mM HEPES pH 7.9, 10 mM KCl2, 1.5 mM MgCl2, 0.34 M sucrose, 0.5% triton, 10% glycerol, 1 mM DTT) for 10 min on ice and washed once in cytoplasmic lysis buffer. Cells were then scraped and lysed using nuclei lysis buffer (50 mM Tris pH 8.0, 5mM EDTA pH 8.0, 1% SDS and 0.5mg/ml proteinase K (Invitrogen, 25530049)) and incubated for 1h at 55°C for protein digestion. Genomic DNA was then precipitated by adding 10% volumes of 3M sodium acetate PH 5.2 and 2.5X volumes of 100% ethanol and incubated at room temperature until DNA is clearly visibly precipitated. The DNA was spooled onto a P1000 pipette tip, washed once in 80% ethanol, air dried and resuspended in 300μl 1X TE buffer. 50 μg of DNA was diluted into 300μl of 1X TE and then sonicated for 13 cycles 10sec on/ 30sec off at 4°C using Bioruptor Plus sonication device (Diagenode) and run on a 2% agarose gel to confirm fragmentation (See supplementary Figure 4A). 25μg of fragmented DNA was then incubated with 50 units of RNase H (NEB, M0297L) for 3h at 37°C, topped up with an additional 50 units of RNase H for extra 3h. Next, 20μg of each sample was immunoprecipitated overnight at 4°C with 10μg S9.6 antibody (Merck, MABE1095) conjugated to 100μl Pierce dynabeads protein G magnetic beads (Thermo, 10004D). Beads were then washed once with 1x DRIP buffer, once with 1x DRIP buffer supplemented with 500mM NaCl, and once in LiCl buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA pH 8.0, 1% NP-40) followed with two washes in 1× TE buffer. To elute DNA from beads, 100μl genomic extraction buffer (1x TE buffer, 0.5% SDS, and 1mg/ml proteinase K) was added, and beads were incubated for 30 min at 55°C. The eluted DNA was subsequently purified via phenol:chloroform:isoamylalcohol (pH 8.0, Sigma P2069) and then ethanol precipitated.   5ng of DRIP DNA from each of three biological replicates from each condition were used for library preparation using the Thruplex DNA-seq kit for Illumina (TAKARA, R400674) according to the manufacturer’s protocol using 12 PCR cycles for IP samples, and 8 PCR cycles for input samples.
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_UT_2
title