ChIP assays were performed using HeLa cells, the H3K4me3 antibody (bs-53034R) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 \u03bcg of antibody per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes

ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 \u03bcg of the H3K4me3 antibody (bs-53034R). The IP\u2019d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively. These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.

A Dot Blot analysis was performed to test the cross reactivity of H3K4me3 antibody (bs-53034R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. The figure shows a high specificity of the antibody for the modification of interest.

Western blot was performed using histone extracts from HeLa cells (15 \u03bcg) and H3K4me3 antibody (bs-53034R) diluted 1:500 in TBS-Tween containing 5% skimmed milk. A molecular weight marker (in kDa) is shown on the left, the position of the protein of interest is shown on the right.

HeLa cells were stained with the H3K4me3 antibody (bs-53034R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were labelled with the H3K4me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.