title: GSM3854338: FigS4B_26415_t20_IP; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: To crosslink cells, 45ml of yeast culture was mixed with 4.2ml of fixation solution (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.5mM EGTA, 1mM EDTA, 30% Formaldehyde) and incubated at 18°C for 30 minutes. The crosslinking reaction was quenched by incubating with 2ml of 2.5M Glycine for 5 min. Fixed cells were harvested, washed with ice-cold PBS and re-suspended in 1ml of ChIP lysis buffer (50mM Hepes-KOH pH 8.0, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1mM PMSF, Roche protease inhibitor). For each ChIP, 10 OD600 units of Saccharomyces cerevisiae cells mixed with 5 OD600 units of Candida glabrata cells were pelleted and re-suspended in 0.3ml of ChIP lysis buffer. Cells were mixed with glass beads and disrupted by FastPrep®-24 (MP Biomedicals, USA). The entire lysis was collected and sonicated at 4°C for 40 minutes using a Bioruptor (Diagenode, Belgium). The cell debris were removed by centrifugation and supernatants containing sheared chromatin with a size range from 100-800bp were adjusted to a final volume of 1ml with ChIP lysis buffer. Extracts were pre-cleared for 1 hour at 4°C with 30µl of Protein G Dynabeads (Invitrogen). 80µl of supernatant was taken as whole cell extract (W) and stored at -80°C. Five µg of anti-PK antibody (Bio-Rad) and 50 µl of Protein G Dynal beads (Invitrogen) was used for immunoprecipitation (6 hours-overnight, rotation at 4°C). The beads were subsequently washed for 5 minutes with the following buffers: 2Ã—ChIP lysis buffer; 3x ChIP high-salt lysis buffer (50mM Hepes-KOH pH 8.0, 500mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1mM PMSF); 2Ã—ChIP wash buffer (10mM Tris-HCl pH 8.0, 0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA, 1mM PMSF) and 1x TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 50mM NaCl). The immunoprecipitated chromatin was eluted by incubation of beads with 120µl of TES buffer (50mM Tris-HCl pH 8.0; 10mM EDTA; 1% SDS) at 65°C for 15 minutes. The supernatants were collected and termed the IP sample. The whole cell extract sample (W) was mixed with 40µl of TES3 buffer (50mM Tris-HCl pH 8.0; 10mM EDTA; 3% SDS). Both samples were de-crosslinked at 65°C overnight. RNA was degraded by incubating with 2-µl of RNAse A (10mg/ml, Roche) at 37°C for 1 hour and protein was subsequently removed by incubation with 10µl of Proteinase K (18mg/ml, Roche) at 65°C for 2 hours. DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research, USA). NEBNext Fast DNA Library Prep set for Ion Torrent, E6270
attributes.tag: GEO Accession
attributes.value: GSM3854338