Preparation of mouse testis protein extracts

For mass spectrometry experiments, nuclear extracts were prepared from mouse testes from 12 to 13 dpp B6 mice (n = 18). Proteins were extracted from nuclei following the Dignam protocol (Dignam et al., 1983).

For analysis of PRDM9 and HELLS expression during mouse spermatogenesis, whole cell extracts were prepared from frozen testes collected from 4, 6, 9, 12, 15 dpp, and adult RJ2 males. Extraction was performed by homogenizing cells with a Dounce homogenizer in 400 mM NaCl, 50 mM Hepes, 1% Triton X-100, 4 mM DTT, complete protease inhibitor, followed by sonication and centrifugation to remove debris.

For PRDM9 and HELLS expression analysis in testes from 22 dpp Hells CTRL and Hells cKO mice, nuclear extracts were prepared. Testes were homogenized in hypotonic buffer (10 mM Hepes, pH 8.0, 320 mM sucrose, 1 mM PMSF, 1x Complete protease inhibitor cocktail EDTA-free (Roche, Cat. Number 11873580001)) in a Dounce homogenizer. After centrifugation (1000xg at 4°C for 10 min), supernatants were collected and used as cytoplasmic fractions. Nuclear fractions were from pellets that were resuspended in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1x Complete protease inhibitor EDTA-free (Roche)), sonicated and centrifuged to remove debris.