title: GSM3229244: FH-Post-meiosis; Mus musculus; ChIP-Seq
library_construction_protocol: Meiotic and post-meiotic cells expressing FLAG-HA tagged endogenous H3.3 were lysed in hypotonic buffer (10 mM Tris-HCl at pH 7.65, 1.5 mM MgCl2, 10 mM KCl) and disrupted by Dounce homogenizer. The cytosolic fraction was separated from the nuclei by centrifugation at 4°C. Nuclei were re-suspended in sucrose buffer (20 mM Tris-HCl at pH 7.65, 15 mM KCl 60 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine) adjusted with high-salt buffer (20 mM Tris-HCl at pH 7.65, 25 % glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 900 mM NaCl) to get a final NaCl concentration of 300 mM. The nuclear-soluble fraction was recovered by centrifugation at 4°C. The pellet containing chromatin fraction was incubated in sucrose buffer containing 1mM CaCl2, and MNase (2.5U/g of cells) 10 min at 37°C. Digestion was stopped with 4 mM EDTA and mononucleosomes were recovered by centrifugation at 4°C. Tagged nucleosomes were immunoprecipitated with anti-FLAG M2-agarose (Sigma), eluted with FLAG peptide (0.5 mg/mL), further affinity-purified with anti-HA antibody-conjugated agarose, and eluted with HA peptide (1 mg/mL). The HA and FLAG peptides were first buffered with 50 mM Tris-Cl (pH 8.5), then diluted to 4 mg/mL in TGEN 150 buffer (20 mM Tris at pH 7.65, 150 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0 0.01% NP40), and stored at −20°C until use. Between each step, beads were washed in TGEN 150 buffer. Nucleosomes were submitted to RNase and proteinase K digestion, and DNA was extracted by phenolchloroform. Libraries were prepared using the Diagenode MicroPlex Library Preparation kit version v.2 02.15, and sequenced on Illumina Hiseq 4000 sequencer as single-end 50 bp reads following Illumina's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3229244