truChIP Chromatin Shearing Kit with Formaldehyde

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	DNA-protein cross-linking was carried out by addition of 11.1% formaldehyde and incubation for 5 min @ 25°C. Cells were lysed by addition of Lysis Buffer A. The lysates were sonicated @ 6°C; and the concentration of sheared DNA was measured by NanoDrop. Four µg of ERα antibody (AER304, mouse monoclonal, Millipore) or mouse non-immune IgG (Millipore) was utilized for IPs.

Protocol tips
DNA-protein cross-linking was carried out by addition of 11.1% formaldehyde and incubation for 5 min @ 25°C. Cells were lysed by addition of Lysis Buffer A. The lysates were sonicated @ 6°C; and the concentration of sheared DNA was measured by NanoDrop. Four µg of ERα antibody (AER304, mouse monoclonal, Millipore) or mouse non-immune IgG (Millipore) was utilized for IPs.

Publication protocol

Subconfluent proliferating LCC9 or T47DCO cells were seeded into 100-mm dishes (6 × 105 cells/dish), incubated for 24-hr, and treated with vehicle or NSC35446.HCl as indicated. ChIP assays were performed using two commercial kits, the truChIP™ Chromatin Shearing Reagent Kit (Covaris, Woburn, MA) and the Magna ChIP™ HiSens Chromatin Immunoprecipitation Kit (Millipore). DNA-protein cross-linking was carried out by addition of 11.1% formaldehyde and incubation for 5 min @ 25°C. Cells were lysed by addition of Lysis Buffer A. The lysates were sonicated @ 6°C; and the concentration of sheared DNA was measured by NanoDrop. Four µg of ERα antibody (AER304, mouse monoclonal, Millipore) or mouse non-immune IgG (Millipore) was utilized for IPs. The DNA was incubated with protein A/G magnetic beads (Millipore) for pre-linking @ 4°C for 2 hr. Five percent of undiluted DNA was set aside as input. DNA corresponding to 7 × 105 cells was incubated with the beads at 4°C overnight. Beads were washed with low stringency IP wash buffer. The DNA was eluted by overnight treatment with Proteinase K and RNase H at 57°C. It was then purified and subjected to qPCR amplification of sequences surrounding the previously identified EREs of the IKKB promoter or the known ERE of the pS2/TFF1 promoter [16] (Supplementary Table S3). Primers and were obtained from IDT Technologies. Thermal cycling conditions were: denaturation at 95°C for 15 sec annealing/extension at 60°C for 1 min, for a total of 45 cycles.

In some experiments, MCF-7 cells were treated ± E2 (10 nM) and ± Fulvestrant (1 µM) for 120 min and assayed to detect ERα at the known EREs of the pS2 or GREB1 [17] promoters. In other experiments, MCF-7 cells were exposed to E2 for 120 min, then treated with vehicle or NSC35446.HCl for 48-hr, and then and assayed to quantify ERα at the pS2 or GREB1 EREs.

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