title: GSM1267081: dcr1∆+vector; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 15 min at room temperature (RT), then quenched with 130 mM glycine for 5 min at RT, harvested by centrifugation, washed twice with TBS (50 mM Tris, pH 7.6,150 mM NaCl), and flash-frozen. Cell pellets 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.  Anti-H3K9me2 antibody (Abcam, ab1220) 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, diluted with 250 ul TE, and treated with 50 ug RNase A at 37°C for 1h. 60 ug glycogen and 5 ul proteinase K (Roche) was added and incubation was continued at 55°C for 1h. 22 ul of 10M LiCl was added and the samples were extracted with phenol/chloroform, and EtOH precipitated. 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 the first 6 nt of each read and are listed in a column for each of the raw files. Each barcode (the 7th nt) should be followed by a T.
attributes.tag: GEO Accession
attributes.value: GSM1267081