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
We labeled nascent DNA by adding EdU (25 µM) for 30 minutes, 1.5 hours after release into S phase. Samples were then collected at 2 hours after release. After two washes in PBS, 8 million cells were harvested by trypsinization. We next lysed the cells in low adhesion tubes by 4 minutes incubation at RT in 500 µl 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 collected the chromatin fraction by centrifugation for 5 minutes at 500 xg. After removal of the supernatant, we next \"clicked\" the cleavable biotin linker (Azide-PEG3-S-S-biotin #BP-22955, tebu-bio) onto EdU by resuspending the lysed nuclei pellet in 300 ul Click-iT buffer, CuSO4 reaction additive, according to manufacturer's instructions (Invitrogen). After 25 minutes incubation at RT in the dark, chromatin was recovered by centrifugation, washed with lysis buffer and processed for MNase, RNase then Proteinase K digestion as described above (native nucleosome extraction). 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). DNA from native nucleosomes was extracted by phenol-chloroform and ethanol precipitation. The DNA pellet was resuspended in 100 ul H2O (input) and biotinylated EdU-labeled fragments were recovered with 33 µl of Streptavidin Magnetic Dynal beads M-280 (#11205D, Invitrogen) by 15 minutes incubation at RT on rotating wheel and subsequent washes according to manufacturer's instructions. Bead-bound DNA was eluted by cleaving the S-S bond with a 1 hour incubation at RT in TE buffer supplemented with 2% beta-mercaptoethanol. Finally, released DNA was purified by phenol-chloroform extraction, EtOH precipitation and resuspended in 20 µl water. 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.