- Highly optimized protocol for chromatin preparation prior to ChIP on histones SDS concentration optimized for the workflow for histones
- Validated for cells and tissue
- Preserves the epitopes
- Validated with the Bioruptor ultrasonicator
- Quality of chromatin sample confirmed by ChIP-seq
Figure 1. Optimal chromatin shearing profile
HeLa cells were fixed with formaldehyde for 8 min and chromatin was prepared according to Diagenode’s Chromatin EasyShear Kit – Ultra Low SDS (Cat. No. C01020010). Samples were sonicated for 5-10-15 cycles of 30” ON/30” OFF as indicated with Bioruptor Pico using 1.5 ml Bioruptor microtubes with caps (Cat. No. C30010016) followed by de-crosslinking and DNA purification. The fragment size was assessed using agarose gel electrophoresis. A 100 bp ladder was loaded as the size standard.
Figure 2. Chromatin precipitation
Sheared chromatin (obtained with the Chromatin EasyShear Kit – Ultra Low SDS) has been used for immunoprecipitation with H3K4me3 and IgG (negative control) antibodies. Quantitative PCR was performed with positive (GAPDH) and negative (TSH2B) control regions. The Figure 2 shows the recovery expressed as % of input (the relative amount of immunoprecipitated DNA compared to input DNA (panel A) and as enrichment fold of positive locus over negative (panel B).