Protein a g agarose salmon sperm dna

Cells were fixed in 1% formaldehyde for 10 minutes at 37°C. Cross-linking was quenched by adding 125 mmol/L glycine. Cells were then washed with cold PBS, harvested and resuspended in SDS lysis buffer containing a protease inhibitor cocktail. Chromatin was sheared by sonication (average length 0.25-1 Kb) and incubated with 60 ml protein A/G agarose/salmon sperm DNA (50% slurry; Millipore) with gentle agitation for 30 minutes. The supernatant was then immunoprecipitated with anti-SOX4 antibody 1:500 or its matched nonimmune crude serum 1:500 (IgG; Diagenode) at 4°C overnight. Protein A/G agarose (60 mL of 50% slurry) was then added and incubated for 1 hour. Pellets were washed and protein-DNA cross-links were reversed by overnight incubation at 65°C with proteinase K. DNA was purified following a conventional phenol–chloroform protocol and eluted in 50 mL water. At least 3 independent Chromatin immunoprecipitation (ChIP) experiments were carried out.

ChIP assays were performed using a ChIP assay kit (Millipore) with modifications. Isolated B cells (5 × 10⁶ cells) were fixed in 1% formaldehyde, and the chromatin was sonicated and pre-cleared by incubation with Protein A/G agarose/salmon sperm DNA (Millipore). The precleared chromatin was immunoprecipitated with antibodies against H3K27me3 (Abcam) overnight at 4°C or mouse IgG monoclonal antibody followed by incubation with Protein A/G agarose/salmon sperm DNA for 1 h. The immunoprecipitates were denatured, and the DNA was purified. The amount of immunoprecipitated DNA was quantified by real-time PCR using SYBR Green and the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The primers used for the PCR analysis of the IL-10 promoter locus are as follows: forward: 5′ to 3′, CCA GGT AGA GCA ACA CTC; reverse: 5′ to 3′ CAG GCT CCT TTA CCC CGA TT.