Histone extraction, Western blot analysis and quantification

Histones were extracted form embryos and cell pellets by acid extraction [85], resuspended in water and quantified by the Coomassie Protein Assay kit (Thermo). Three microgram of histones was diluted in SDS-loading buffer and run on a 12 % SDS-polyacrylamide gel electrophoresis, followed by Western blotting onto Hybond ECL membrane (Amersham) according to the standard protocols. Membranes were saturated with 3 % skimmed milk powder in Tris-buffered saline with Tween (TBST) and incubated overnight at 4 °C with the primary antibody diluted in the same buffer. The primary antibodies were the same as above, with the addition of anti-Histone H3 C-terminal (#07-690, Millipore), and were diluted 1/2000, excepted for anti-Histone H3 C-terminal, diluted 1:50,000. They were washed with TBST and incubated 1 h at RT with the appropriate peroxidase-conjugated secondary antibody. The secondary antibodies were anti-mouse-peroxidase (#115-035-003) and anti-rabbit-peroxidase (#115-036-003) from Jackson Immunoresearch, diluted 1:10,000. Signal detection was performed using the Clarity ECL kit (Biorad) and Biorad Chemidoc MP imaging system. Membranes were then stripped according to the manufacturer’s protocol and re-probed with the antibody against histone H3 as a loading control. Integrated pixel intensity was measured with ImageJ [84] for each band and the respective background signal was subtracted. Signals were normalised to the loading control (histone H3), and the fold difference to the control cell type (CEF) was calculated.