ChIP-seq of MYB in adenoid cystic carcinoma (ACC) ACCX11 cells
ChIP-seq of MYB in adenoid cystic carcinoma (ACC) ACCX11 cells
5x10^6 cultured cells were used for immunoprecipitation. To crosslink proteins to DNA, 540 Âµl of 37% formaldehyde was added to cells in 20 ml culturing medium and incubated for 10 minutes at room temperature. 2 ml of 10X glycine was added and incubated for 5 min at room temperature. Cells were pelleted at 500 x g at 4 degrees for 5 minutes and washes in ice-cold PBS + proteinase inhibitors. Chromatin extraction, digestion, and digestion was performed using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) according to manufacturer's instructions. Chromatin was digested using micrococcal nuclease (MNase) prior to chromatin immunoprecipitation (ChIP) with anti-MYB antibody (recombinant anti-MYB phosphoS11, Abcam). Following ChIP, chromatin was eluted from from Antibody/Protein G Magnetic Beads and reverse crosslinked. DNA was purified by the use of spin columns.  Library construction was performed by Novogene UK (Cambridge, UK). Purified DNA (extracted and digested using SimpleChIP Enzymatic Chromatin IP Kit, Cell Signaling) was supplied for sequencing. Novogene UK constructed DNA libraries as follows: DNA fragments were repaired, A-tailed, and further ligated using Illumina adapter. The final DNA library was obtained by size selection and PCR amplification.