title: new H3.3 2.0h (HIRA WT)
description: Protocols: To selectively track newly synthesized H3.1- and H3.3-SNAP, we quenched the SNAP-tag of parental (old) histones by exposing G1/S cells to 10 µM SNAP-Cell Block (S9106S, New England Biolabs) for 30 minutes, followed by 2 hours chase (cells incubated in media without SNAP-Cell Block but containing 2 mM thymidine). Cells were then released into S phase by three PBS washes and incubation in DMEM complete medium supplemented with 24 µM 2-Deoxycytidine. 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). We prepared SNAP-Capture magnetic beads (S9145S, New England Biolabs) by washing and coating for 1 hour in coating buffer (PBS, 2.5% BSA, 0.05% Tween 20). We mixed 10 ul of SNAP-Capture magnetic beads with the native nucleosomes (input) and incubated overnight at 4°C on a rotating wheel. We collected beads with a magnetic rack, discarded the supernatant and washed beads in 1 mL, twice with wash buffer 1 (10 mM Tris-HCl pH 8, 140 mM NaCl, 1% Triton X-100, 0.5% NP40, 0.1% SDS), twice in wash buffer 2 (10 mM Tris-HCl pH 8, 360 mM NaCl,1% Triton X-100, 0.5% NP40, 0.1% SDS), twice in wash buffer 3 (10 mM Tris-HCl pH 8, 250 mM LiCl, 0.5% Triton X-100, 0.5% NP40) and twice in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were finally resuspended in 20 uL TE and DNA was released from bound nucleosomes by RNAse A (1 ug/ul) and subsequent proteinase K digestion (2 ug/ul and supplementation to 2% SDS). Beads were removed by magnetic separation and DNA was extracted using Agencourt AMPure XP Beads (A63880, Beckman Coulter) according to the manufacturer's instructions, and eluted in 20 ul 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.
attributes.tag: ENA first public
attributes.value: 2022-03-31
attributes.tag: ENA last update
attributes.value: 2022-03-31
attributes.tag: External Id
attributes.value: SAMEA9273561
attributes.tag: INSDC center alias
attributes.value: Nuclear Dynamics Unit UMR3664 CNRS/IC Institut Curie - Centre de Recherche
attributes.tag: INSDC center name
attributes.value: Nuclear Dynamics Unit UMR3664 CNRS/IC Institut Curie - Centre de Recherche
attributes.tag: INSDC first public
attributes.value: 2022-03-31T00:17:17Z
attributes.tag: INSDC last update
attributes.value: 2022-03-31T00:17:17Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10619:new H3.3 2.0h (HIRA WT)
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HeLa
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: cervical adenocarcinoma
attributes.tag: genotype
attributes.value: H3.3-SNAP-HA GFP CRISPR/Cas9 KO
attributes.tag: organism part
attributes.value: uterine cervix
attributes.tag: sample name
attributes.value: E-MTAB-10619:new H3.3 2.0h (HIRA WT)
attributes.tag: sex
attributes.value: female