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|The sheared cross-linked chromatin was immunoprecipitated (IP) using ChIP-validated antibodies to acetyl-histone H3, acetyl-histone H3- Lys9 (H3K9), trimethyl-histone H3-Lys9 (Upstate Biotechnology), HDAC1 and RNA Pol II (Santa Cruz Biotechnology). Each IP reaction consisted of 125 μl of chromatin + 375 μl of dilution buffer with protease inhibitors + 20 μl of protein G magnetic beads + 5 μg of primary antibody. The IP reactions were incubated at 4° C overnight with rotation. IgG from the same species as the primary antibodies served as negative controls. Magnetic beads were separated using a magnetic separator (Biolabs) and the supernatant was discarded. The Protein G magnetic beads-antibody-chromatin complex was incubated with a series of wash buffers provided in the Magna ChIP G tissue Kit: one time each for 5 min each wash on a rotating platform followed by magnetic clearance and careful removal of the supernatant fractions: 500 μl low salt immune complex wash buffer, 500 μl high salt immune complex wash buffer, 500 μl LiCl immune complex wash buffer, 500 μl TE buffer.|
|-It is highly critical that the chromatin is of appropriate size and concentration.|
|-For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
|-Once in solution, store 1M DTT at -20°C.|