H3k36me3 antibodies

Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10 million cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x 10 min (30 s on; 30 s off). 67 μl of chromatin (1 million cells) was incubated with 229 μl dilution buffer, 3 μl protease inhibitor cocktail and 0.5-1 μg of H3K27ac, H3K4me3, H3K4me1, H3K27me3, H3K9me3 or H3K36me3 antibodies (Diagenode) and incubated overnight at 4°C with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 min at 4°C. Beads were washed with 400μl buffer for 5 min at 4°C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 min. Supernatant was collected, 8 μl 5M NaCl, 3μl proteinase K were added and samples were incubated for 4 hr at 65°C.Finally samples were purified using QIAGEN; Qiaquick MinElute PCR purification Kit and eluted in 20 μl EB. Detailed protocols can be found on the Blueprint website (http://www.blueprint-epigenome.eu/UserFiles/file/Protocols/Histone_ChIP_May2013.pdf).