GSM3760088: WT_H3ac; Mus musculus; ChIP-Seq
aNSCs were isolated from adult mice (between 2 and 4 months old) as previously described (Walker and Kempermann;2014) and expanded In-vitro. 2x106 Cells were cross-linked by adding formaldehyde 1% for 10 min at room temperature, quenched with 0.125 M Glycine for 5 min at room temperature, and then washed three times in phosphate-buffered saline (PBS) before freezing. Pellets were suspended in 0.25 ml SDS lysis buffer (50 mM HEPES-KOH, 140 mM NaCl, 1mM EDTA , 0.1% Triton X-100, 0.1% Sodium Deoxycholate, 1% SDS, 10 µM NaB and protease inhibitors), incubated on a rotator for 30 min at 4oC, sonicated for 20 min at 4°C, and then centrifuged at 14,000 rpm for 10 min at 4°C. Supernatants were diluted 10-fold with ChIP dilution buffer (1% Triton X-100, 2mM EDTA, 20mM Tris-HCL, 150 mM NaCl, 10 µM NaB and protease inhibitors) (25 µl were kept as input) and incubated overnight in gentle rotation at 4°C with 4 µg of antibody. The following antibodies were used: rabbit anti-SP1 (Millipore), rabbit anti-acetyl-Histone 3 (Millipore), mouse anti-TRRAP (Euromedex) and rabbit anti-acetyl-Histone 4 (Millipore). After that, 40 µl of preblocked protein-G-conjugated magnetic beads (DYNAL, Thermo Fischer) were added and incubated for 2h in a rotator at 4°C. The immunoprecipitated complexes were washed three times in low-salt wash buffer (0.1% SDS,1% Triton X-100, 2mM EDTA, 20mM Tris-HCL, 150 mM NaCl), one time in high-salt wash buffer (0.1% SDS,1% Triton X-100, 2mM EDTA, 20mM Tris-HCL, 500 mM NaCl) and one time in TE buffer. The complexes were eluted by adding 0.2 ml of Elution buffer (TE 1x, 1 % SDS, 150 mM NaCl, 5mM DTT) for 30 min in rotation at room temperature. The de-crosslinking was performed at 65 °C overnight. De-crosslinked DNA was purified using QiaQuick PCR Purification Kit (Quiagen) according to the manufacture's instruction. Approximately 10 ng of purified ChIP DNA were end-repaired, dA-tailed, and adaptor-ligated using the TruSeq ChIP Sample Preparation Kit (illumina) following the manufacturer's instructions. The size of the library was checked using Fragment Analyzer (Agilent) and the library was sequenced on the NextSeq500 platform (illumina).