Histone acid extraction

Starting material was a pellet of 50-100M cells (washed once with cold PBS) or a flash-frozen tissue homogenate in liquid nitrogen using a ceramic mortar grinder. Cells were washed first in 10ml of buffer I (10 mM TrisHCl pH 8, 10 mM MgCl₂, 0.4M Sucrose). After 5min incubation, samples were centrifuged at 8.000g for 20min at 4°C and supernatant was removed. The resulting pellet was resuspended in 1.5ml of Buffer II (10 mM TrisHCl pH 8, 10 mM MgCl₂, 0.25M Sucrose, 1% Triton X-100, 1% Igepal Ca-630) and incubated 15min on ice. In specific cases, cells at this stage were broken using a 2ml Dounce homogenizer (with Pestle B) or with a 20G syringe. Then samples were centrifuged at 15.000g for 10min at 4°C and supernatant was removed. The resulting pellet was then slowly resuspended in 300μL of Buffer III (10 mM TrisHCl pH 8, 2 mM MgCl₂, 1.7M Sucrose, 1% Triton X-100) and then resulting resuspended nuclei were layered on top of another 300μL of Buffer III. Sample was centrifuged at 20.000g for 1h at 4°C and supernatant was removed, resulting in a nuclear pellet ready for acid histone extraction. All buffers were supplemented with spermidine (1:1000), beta-mercaptoethanol (1:1000), protease inhibitors (1x cOmplete cocktail Roche #11697498001, 1mM PMSF, 1:2000 Pepstatin), phosphatase inhibitors (1x phoSTOP cocktail Roche #4906845001) and deacetylase inhibitors (10mM Sodium butyrate).

For samples processed using a high-salt + HCl extraction protocol^101,102, the pellet was resuspended in 500μL of High Salt Extraction Buffer (20 mM TrisHCl pH 7.4, CaCl₂ 1M and protease, phosphatase and deacetylase inhibitors, same as above). Sample was incubated on ice for 30min and then pure HCl has added to a final 0.3N concentration (12.82μL to the initial 500μL). Samples were incubated for at least 2h on a rotor at 4°C and then centrifuged at 16.000g for 10min at 4°C to remove cellular/nuclear debris. The resulting supernatant containing solubilized histones was transferred to a clean 1.5ml tube and Trichloroacetic Acid (TCA) was added drop-wise to 25% final concentration (171μL TCA to an approximate initial 513μL sample) and left overnight at 4°C to precipitate histones. Samples were then centrifuged at 20.000g for 30min at 4°C and the supernatant removed. The pellet was then washed twice with 500μL of cold acetone and then dried for 20min at room temperature. Finally, clean histone pellets were resuspended in 30-50μL of ultrapure water. Protein concentration in the sample was measured using BCA and extraction was examined using an SDS-PAGE protein gel with Coomassie staining.

For samples processed using H₂SO₄¹⁰², the protocol was exactly the same except that 400μL 0.4N H₂SO₄ (freshly diluted) was used instead, with a similar incubation time of at least 2h at 4°C.