GSM7519222: FLAG; TetR-SET; Suv39x: ChIP-seq (A); Saccharomyces cerevisiae; ChIP-Seq
Cells were fixed in the presence of 1% formaldehyde for 15 minutes at room temperature with gentle mixing on a moving platform.  For FLAG3X-Suv39x and Sir3-FLAG3X-SET immunoprecipitations, cells were fixed in the presence of 1.5 mM ethylene glycol bis(succinimidyl succinate) (EGS) for 30 minutes at room temperature and then fixed in the presence of 1% formaldehyde for 30 minutes at room temperature.  Crosslinking reactions were quenched in the presence of 125 mM glycine.  Crosslinked cells were pelleted, washed twice in cold tris-buffered saline (TBS), once in cold water, and stored at -80oC.  Samples were sonicated in a cold room using a tip sonicator (Sonics VCX 500; set at 30% amplitude) for a total of 80 seconds, with five-minute incubations on ice after every 20 seconds of sonication.  Samples were further sheared by sonication using a Q800R3 sonicator (Qsonica; set at 20% amplitude).  Samples were sonicated for a total of 15 minutes, with 15-second incubations without sonication following every 15 seconds of sonication. Libraries were prepared by ligating Y-adapters encoding unique barcode sequences (Wong, Jin, and Moqtaderi, 2013) to immunoprecipitated DNA.  Libraries were amplified by PCR for 18 cycles.  Amplified DNA 150-500 bp in length was purified using a gel extraction kit (Omega Bio-tek) and SPRIselect beads (Beckman Coulter).