Chipab trimethyl histone h3 lys4

The following antibodies were employed: histone H3 antibody (Abcam, ab1791), Histone H3 (di methyl K9) antibody (Abcam, ab1220), ChIPAb+ Trimethyl-Histone H3 (Lys4) (Millipore, 17-614), Anti-acetyl-Histone H4 antibody (Millipore, 06-866), RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (Abcam, ab5408), anti-histone H3 monomethyl K4 antibody (Abcam, ab8895), anti-histone H3 acetyl K9 antibody (Abcam, ab4441), anti-histone H3 trimethyl K9 antibody (Abcam, ab8898), anti-trimethyl-histone 3 (Lys27) (Millipore, 07-449), anti-histone H3 (acetyl K27) antibody (Abcam, ab4729), and anti-Flag M2 antibody (Sigma, F1804).

ChIP was carried out using the EZ-Magna ChIP A Chromatin Immunoprecipitation Kit (Millipore). A total of 20 × 10⁶ CD4⁺ T cells were fixed in 1% formaldehyde (Fisher Scientific). Glycine buffer was added to quench any remaining formaldehyde. Cells were then lysed and sonicated using a Diagenode bath sonicator at high setting with 30-s pulses over a 5-min period. Immunoprecipitation was carried out using the following antibodies: anti-acetyl-histone H3 (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys4) (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys27) (Millipore), anti-Histone H3 (di methyl K9) (Abcam), and rabbit IgG (Millipore). Real-time PCR analysis was carried out using the MJ Opticon 2 thermo cycler (MJ Research) and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Cycling conditions were set up according to the manufacturer's instructions; annealing temperature for all primers was 55°C with 40 cycles of amplification. For ChIP primers and statistical analysis, see Supporting Information Method 2.