Illumina NovaSeq 6000 paired end sequencing; De novo deposition of the histone H3 variants H3.1 and H3.3 during DNA replication
De novo deposition of the histone H3 variants H3.1 and H3.3 during DNA replication
In parallel we probed total H3.1- and H3.3-SNAP in G1/S cells at the time of release (0.0h). Samples were collected after 2.5 hours, without previously adding SNAP-Cell Block to the medium (no quench). We used HeLa cells stably expressing H3.3-SNAP-HA or H3.1-SNAP-HA, and derived HIRA knockout cell lines (HIRA CRISPR/Cas9 KO) or HIRA wild-type control cells (GFP CRISPR/Cas9 KO) as described in Ray-Gallet et al. 2019. We cultured cells in DMEM complete medium with D-Glucose, L-Glutamine and Pyruvate, supplemented with 10% fetal calf serum, 100 U/mL Penicillin and 100 µg/mL Streptomycin. Cells were grown in 14 cm Petri dishes and synchronized in G1/S via double thymidine block: subsequent exposure to 2 mM thymidine (16-18h), 24 µM 2-Deoxycytidine (6h) and 2 mM thymidine (18h). All cell lines tested negative for mycoplasma contamination. We harvested synchronized cells by trypsinization and counted them. Four million cells were collected, washed with PBS and pelleted by centrifugation in low adhesion tubes. We processed one million cells at a time, resuspended the cell pellet in 100 uL lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1% Triton X-100, 0.5% NP40 and cOmplete EDTA-free protease inhibitor cocktail by Roche) and incubated for 4 minutes at room temperature. After centrifugation (5 minutes, 500 xg) we eliminated the cytosolic fraction and resuspended the pelleted chromatin fraction in 25 ul lysis buffer. We then performed a micrococcal nuclease digestion with 3U MNase (#EN0181, ThermoScientific) and 8 minutes incubation at 37°C. We stopped digestion by addition of EGTA to 20 mM. After 10 minutes incubation on ice we centrifuged 10 minutes at 10000 xg. We resuspended the pellet with the supernatant to thoroughly extract soluble nucleosomes and proceeded to a second 10 minutes 10000 xg centrifugation. We recovered the supernatant containing the native nucleosomes in low adhesion tubes, and added 5 volumes of binding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 0.5% BSA and cOmplete EDTA-free protease inhibitor cocktail by Roche). After checking purified DNA by Bioanalyzer profile, sequencing libraries were prepared by the Next Generation Sequencing (NGS) platform at Institut Curie using the Illumina TruSeq ChIP kit.