GSM6049423: ChIPseq_Input_rrp6_t1_rep3; Saccharomyces cerevisiae; ChIP-Seq
RNA was extracted with MasterPure RNA extraction kit from Lucigene  The DNA was purified with phenol, precipitated with ethanol and sodium acetate at -20 °C, washed with cold 70% ethanol, and resuspended in 30 µl TE buffer. RNA-Seq: First rRNA was depleted  from total RNA with Illumina ribozero Gold kit. Then stranded RNA library was generated with NEBnext Ultra Directional RNA Library Prep kit for illumina.  MNase and ChIPSeq-Seq:  100 ml culture per S. cerevisiae sample and 30 ml of S. pombe culture was crosslinked using 1% formaldehyde for 15 minutes at room temperature. Formaldehyde was quenched by 0.125 M glycine for 5 minutes. Then cells were washed three times with cold TBS, flash-frozen in liquid nitrogen and stored at -80 °C.  Frozen cell pellets were resuspended in zymolyase solution (1 M sorbitol, 50 mM Tris-HCl pH 7.5, 1% beta-mercaptoethanol, 0.1 U/µl zymolyase). The zymolyase digestion proceeded at +37 °C for 30 min (S. cerevisiae) or 90 min (S. pombe). Spheroplasts were isolated by centrifugation at 6000xg for 10 minutes at +4 °C. Spheroplasts were resuspended in NP buffer (10 mM Tris-HCl pH 7.5, 1 M sorbitol, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40 (Tergitol), 1% beta-mercaptoethanol, 0.5 mM spermidine, 1% yeast protease inhibitor cocktail). The suspensions were pre-warmed and then 0.5 U/µl of MNase for S. cerevisiae and 2 U/µ