GSM1545361: SPY2765_ago1Δ,clr4re_H3K9me2; Schizosaccharomyces pombe; ChIP-Seq
Fixed cells were resuspended in 500 ul lysis buffer (50 mM Hepes-KOH, pH 7.5, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors) and disrupted by bead beating (Mini Beadbeater, Biospec products) for 6x30 sec with 1 ml 0.5 mm glass beads. Tubes were punctured and the flow-through was collected in a new tube by centrifugation. After sonication for 3x20 sec at 40% amplitude (Branson Digital Sonifier), the extract was centrifuged (Eppendorf 5415R) for 15 min at 13000 rpm. Antibody was pre-incubated with washed Dynabeads Protein A, and for each immunoprecipitation, 2 ug antibody coupled to 30ul beads was added to 400 ul soluble chromatin, and lysis buffer was added for a final volume of 600 ul. Samples were rotated for 2h at 4°C, the beads were collected on magnetic stands, and washed 3 times with 1 ml lysis buffer and once with 1 ml TE, and eluted with 100 ul pre-heated buffer (50 mM Tris pH 8.0, 10mM EDTA, 1% SDS) at 65°C for 15 min. The eluate was collected and 150 ul 1xTE/0.67% SDS was added. Input and immunoprecipitated samples were incubated overnight at 65°C to reverse crosslinks, and treated with 50 ug RNase A at 37°C for 1h. DNA was cleaned up with QIAquick PCR Purification Kit (Qiagen). Libraries for Illumina sequencing were constructed following the manufacturer's protocols, starting with ∼5 ng of immune-precipitated DNA fragments. Each library was generated with custom-made adapters carrying unique barcode sequence at the ligating end (Wong and Struhl, 2011). Barcoded libraries were mixed and sequenced with Illumina HiSeq2000. Barcodes are 6/8/9 nt in length and should be followed by a T.