Homo sapiens
Protocols: MCF-10-2A TetOn-CENPA-FLAG-HA cells stably expressing a dominant-negative p53 vector (p53-DN) were cultured for 24 days in the absence (no Dox) or presence of Dox (10 ng/mL) to induce basal or high CENP-A expression, respectively. Culture medium (+/- Dox) was renewed every 2-3 days. We harvested cells at days 10 and 24 post-induction. For each IP, 2-5 million cells were harvested and processed in aliquots of 1 million cells per tube by resuspending cell pellets in 100 µL ice-cold lysis buffer for a 4-min incubation at room temperature. We prepared antibody-coupled Protein A Dynabeads by blocking for 3h at room temperature in 1 mL blocking buffer, washing once with PBS-T and incubating for 1h at room temperature in 200 µL PBS-T with 5 µg anti-H3.3 (Active Motif, 91191), 10 µg anti-CENP-A (Cell Signaling, 2186), or 10 µg control IgG (Abcam, ab46540) antibodies. After a wash in binding buffer, we added the antibody-coupled beads to the chromatin and incubated overnight at 4°C on a rotating wheel. The next day, beads were recovered using a magnetic rack and sequentially washed with appropriate buffers. Beads were treated with RNase A and proteinase K in the presence of 2% SDS. DNA was purified using AMPure XP beads (Beckman Coulter, A63880) as per the manufacturer's protocol and eluted in nuclease-free water. Quality and concentration were assessed using a TapeStation 4200 (Agilent) and a Qubit 3.0 fluorometer (Invitrogen). Sequencing libraries were prepared with the Illumina TruSeq ChIP kit.
ENA first public
2026-04-21
INSDC center name
Institut Curie, Université PSL, Sorbonne Université, CNRS, UMR 3664 Nuclear Dynamics, Chromatin Dynamics lab
INSDC status
public
Submitter Id
E-MTAB-15579:IP-H33_MCF_1DOX_D10
broker name
ArrayExpress
cell line
MCF10-2A
cell type
Breast cell
collection date
not collected
common name
human
developmental stage
Adult
disease
Breast fibrocystic disease
genotype
p53-DN TetOn-CENPA-FLAG-HA
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
Mammary gland
sample name
E-MTAB-15579:IP-H33_MCF_1DOX_D10
scientific_name
Homo sapiens
sex
female