GSM7773465: 180525L05_exp180222_MS80_SCGE_anti-Rpb1; Saccharomyces cerevisiae; Nakaseomyces glabratus; ChIP-Seq
S. cerevisiae cultures were combined with spike-in cultures (C. glabrata or S. pombe) and immediately fixed  with formaldehyde at a final concentration of 1% for 15 minutes. Cells were then quenched with 0.125 M glycine (5 minutes), washed twice in cold PBS, pelleted, snap-frozen, and stored at −80 °C. Pellets were thawed and lysed in 300 µl FA lysis buffer (50 mM HEPES–KOH pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF) with ∼1 ml ceramic beads on a Fastprep-24 (MP Biomedicals). The entire lysate was then collected and adjusted to 1 ml before sonication with a 1/8” microtip on a Q500 sonicator (Qsonica) for 8-16 minutes (cycles of 10 seconds on and 20 seconds off). The sample tube was held suspended in a −20 °C 80% ethanol bath to prevent sample heating during sonication. Cell debris was then pelleted, and the supernatant was retained for ChIP or input. For each ChIP reaction, 20 µl Protein G Dynabeads (Invitrogen) were blocked (PBS + 0.5% BSA, incubate 40 minutes at room temperature), pre-bound with 5 µl of antibody in PBS (incubate 40 minutes at room temperature), and washed 2x with PBS before being incubated with supernatant (4°C overnight). Dynabeads were then washed (5 minutes per wash) 2x in FA lysis buffer and 3x in high-salt FA lysis buffer (50 mM Hepes-KOH pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF) and DNA was then eluted in ChIP elution buffer (50 mM TrisHCl pH 7.5, 10 mM EDTA, 1% SDS) at 65 °C for 20 minutes. 15 µl of input was mixed directly with 115 µl of ChIP elution buffer for input samples. Crosslinks were then reversed (65 °C, 5hr) before treatment with RNAse A (37 °C, 1 hour) and then Proteinase K (65 °C, 2 hours). DNA was then purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Indexed sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep kit (NEB Cat # E7645), pooled, and then sequenced by paired-end (2x150bp) Illumina sequencing. spike-in normalised ChIP-seq