title: GSM3986394: H3K27me3_Old; Nothobranchius furzeri; ChIP-Seq
library_construction_protocol: Tissue collection was performed in fishes euthanized with MS-222 and cooled on crushed ice. Skeletal muscle tissues 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-H3K9ac (Abcam), mouse anti-H3K27me3 (Abcam). 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 (Qiagen) according to the manufacture's instruction.
identifiers.namespace: GEO
identifiers.id: GSM3986394