title: GSM2060774: H3K36Me3_ChIPseq_LNCaP; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP assays were carried out according to the manufacturer’s protocol (Upstate Biotechnology). Briefly, ~ 2 x 10^6 cells were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 minutes at 37 °C. The cells were washed twice with ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin and 1µg/ml pepstatin A), harvested and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths of 200 to 500 basepairs. The complexes were immunoprecipitated with antibodies specific for Histone H2AZ (Active motif # 39113), acetylated Histone H2AZ (Abcam #ab18262), H3K4Me1 (Active Motif #39297), H3K27ac (Active Motif #39133), H3K9K14ac (Millipore #06-599.) and H3K36Me3 (Abcam ab9050-100). Ten µl of antibody was used for each immunoprecipitation. No antibody controls were also included for each ChIP assay and no precipitation was observed by quantitative Real-Time PCR (qPCR) analysis. Input samples were processed in parallel. The antibody/protein complexes were collected by either salmon sperm DNA/protein A agarose slurry or Protein A/G PLUS agarose beads (Santa Cruz sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3 and samples were treated with proteinase K for 1 hour and DNA was purified by phenol/chloroform extraction, ethanol precipitation and resuspended in 30 ul H2O. TruSeq ChIP sample prep kit
identifiers.namespace: GEO
identifiers.id: GSM2060774