GSM2066440: HCC1954 LCC2 PolII; Homo sapiens; ChIP-Seq
Cells were grown to 90–95% confluence, fixed with 1% formaldehyde for 15 min at room temperature followed by addition of glycine (1.25mM) for 10 minutes to stop cross linking. Nuclei were prepared and sonicated in lysis buffer (150 mM NaCl, 1% Triton X-100, 20 mM Tris pH 8.0, 0.1% SDS) using a Bioruptor to fragment chromatin to less than 500 base pairs (bp). Chromatin was pre-cleared with 35 μl Protein-G Dyna magnetic beads, and then immunoprecipitated with 5 μg of antibody over night at 4 °C. The chromatin antibody mix was incubated with 35 μl protein G-conjugated Dyna beads for 4 h at 4 °C, washed three times with wash buffer (1% Triton X-100, 50 mM Tris pH 8.0, 10% glycerol) with increasing concentrations of NaCl (150 mM, 300 mM and 400 mM), and two times with Tris-EDTA buffer. DNA was eluted in 1% SDS in Tris-EDTA buffer for 45 min at 37 °C, crosslinking was reversed overnight at 65 °C, and DNA was purified using Qiagen columns. For each ChIP, one fifth of the cells from 15cm plates were used.
GEO Accession
GSM2066440