title: GSM2966295: KDM3A_shKDM3A_ChIPSeq; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked using 1% formaldehyde for 10 min at 298 K. Formaldehyde was removed and cells were incubated with 125 mM glycine for 5 min at 298 K. Nuclear extracts were collected and sonicated to obtain 300 bp chromatin fragments using the Covaris S2 ultrasonicator (Covaris, Woburn, MA). 100 µg of chromatin was incubated with 5 µg of AR (PG21, 06-680, Sigma EMD Millipore, Darmstadt, Germany), 5 µg of KDM3A (A301-538A, Bethyl Laboratories, Montgomery, TX), 2 µg of H3K9me1 (ab9045, Abcam, Cambridge, MA), or 2 µg of H3K9me2 (07-441, Sigma EMD Millipore, Darmstadt, Germany) antibodies overnight at 277 K followed by incubation with 30 µl of protein A/G beads for 4 hours. After four washes, crosslinking was reversed. Chromatin was digested with ribonuclease A followed by proteinase K. Then the DNA was purified using spin columns. The size of the DNA was confirmed by a bioanalyzer (Agilent Biotechnologies, Savage, MD). The purified DNA library was sequenced using an Illumina HighSeq2000 at the Sanford-Burnham Medical Research Institute at Lake Nona, National Genome Library Core Facility.