title: Distribution of total histone variants H3.1 and H3.3 in pluripotent mouse embryonic stem cells (mESCs) and in vitro differentiated neuronal precursor cells (NPCs)
design: Distribution of total histone variants H3.1 and H3.3 in pluripotent mouse embryonic stem cells (mESCs) and in vitro differentiated neuronal precursor cells (NPCs)
library_construction_protocol: We induced synthesis of H3-SNAP-Tag in ESCs and NPCs by adding 1 μg/ml doxycycline before cell collection for 48h. We then collected the samples as described in Gatto et al., 2022, Forest et al., 2023. 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 cultured all cells at 37°C in 5% CO2: KH2 mESCs (Beard et al., 2006) on gelatinized feeder-free tissue culture plates in ESC media (Dulbecco's modified Eagle's medium supplemented with GlutaMax, Pyruvate and 4,5 g/L D-Glucose (Thermo Fisher Scientific), 15% calf fetal serum (Eurobio), 1000 U/ml penicillin/streptomycin, 1X MEM non-essential amino acids, 125 μm beta-mercaptoethanol supplemented with 1000 U/ml LIF (Millipore) and 2i inhibitors, which include 1 μM MEK1/2 inhibitor (PD0325901) and 3 μM GSK3 inhibitor (CHIR99021). To generate NPCs, we cultured mESCs cells in NPC media (ESC media (without LIF and 2i) supplemented with 1 μM Retinoic Acid (RA), 1x N-2 Supplement (Gibco) and 1x B27 Supplement (Gibco) for 4 days, and 3 additional days in the same NPC media supplemented with 10 ng/ml of FGF (Peprotech) and 20 ng/ml of EGF.  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.