CENP-A ChIP-seq of MCF10-2A cells to study CENP-A localization following reversal from high to basal CENP-A expression
CENP-A ChIP-seq of MCF10-2A cells to study CENP-A localization following reversal from high to basal CENP-A expression
MCF-10-2A TetOn-CENPA-FLAG-HA cells stably expressing a dominant-negative p53 vector (p53-DN) were cultured for 41 days in the absence (no Dox), presence of Dox (10 ng/mL) or with interrupted induction with Dox (10ng/mL Dox for 24 days followed by 17 days without Dox) to induce basal or high CENP-A expression. Culture medium (+/- Dox) was renewed every 2-3 days. We harvested cells at day 41 post-induction. For each IP, 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.