Chromatin immunoprecipitation (ChIP) for histone modifications and RNA polymerase II

Sort purified lymphocytes from LNs and spleen (~5 x 10⁶ cells total) were fixed in 1% formaldehyde, resuspended in ChIP lysis buffer (1% v/v SDS, 10mM EDTA, 50 mM Tris-HCl) and sonicated to generate 200–1000 base pair fragments. Samples were then precleared using Protein A-agarose/salmon sperm DNA (Millipore, 16–157), split into 5 and incubated overnight with either 5 ug anti-H3K27me3, 3 ug anti-H3K4me3 or 4 ug anti-RNA polymerase II (all Invitrogen). A no antibody control and a total input positive control were also included. After washing, all samples (except the ‘total input’) were incubated with Protein A-agarose/salmon sperm DNA with rotation for 1 hour followed by a series of washes in low salt, high salt, lithium chloride, and TE buffers. DNA was eluted before crosslink reversal with 0.2M NaCl at 66°C overnight, followed by protein digestion with proteinase K (Promega). Immunoprecipitated DNA was extracted by phenol: chloroform:isoamyl (25:24:1) extraction and resuspended in HPLC water. For analysis, real-time PCR was used to measure the levels of ChIP-DNA, such that resulting cycle threshold (Ct) values were converted to copy number (#copies = 10⁵/2^Ct-17) and samples were normalised to their corresponding total inputs with background subtraction (no-antibody control).