GSM9701574: Pli1-12Pk_Input; Schizosaccharomyces pombe; ChIP-Seq
Logarithmic fission yeast cultures of untagged control, Pli1-12Pk or Nse2-12Pk were mixed with a calibrator – an aliquot of logarithmic budding yeast culture Ku70-cMyc. Immunoprecipitation of Pli1-12Pk- or Nse2-12Pk-bound DNA was then performed. Obtained samples were subjected to next generation sequencing (NGS) and signals from the IP/INPUT ratio were normalized to the budding yeast calibrator. In details, 100 mL of exponential culture (OD600 ~1) of untagged strain, Pli1-12Pk and Nse2-12Pk were mixed with 10 mL of budding yeast exponential culture Ku70-cMyc, used as a calibrator and then crosslinked with 1% formaldehyde (Sigma-Aldrich, F-8775) for 15 min. Formaldehyde crosslinking was quenched with 2.5 M glycine and cells were washed with 1x TBS. Next, samples were frozen in liquid nitrogen and cells were lysed by bead beating in FA-lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA with 1 mM PMSF and protease inhibitors (Sigma-Aldrich, P8215)). Chromatin sonication was done in a Bioruptor Pico (Diagenode) using Easy mode 10 cycles of 30 s ON and 30 s OFF. Immunoprecipitation was carried out overnight: 300  µL of extract was incubated on the wheel with anti-Pk antibody (Bio-Rad, MCA1360G) at 1:150 concentration. 20 µL of extract was preserved as INPUT fraction. Next morning Protein G Dynabeads (Invitrogen, 10003D) were added for 1 h, stringency washes to remove unspecific proteins were done and immunoprecipitated complexes were decrosslinked overnight at 65° C. Then samples were digested with RNAse A (Thermo Scientific, EN0531) for 30 min at 37oC and subsequently with proteinase K at 65oC for 1h. The associated DNA was then precipitated with isopropanol/glycogen/3M potassium acetate for 1h at -20oC. Precipitated DNA was centrifuged for 20 min at 4oC and then washed with cold 70% ethanol. Finally, DNA pellet was resuspended in 30 µL of Nuclease-Free Water (Sigma-Aldrich, W4502). DNA concentration was determined by Qubit4 Fluorometer (Invitrogen) using Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and quality control qPCR was done on 10x dilution of obtained DNA suspension with SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, 1725274, primers listed in Table S2). Samples were then send for DNA libraries preparation and next generation sequencing (Novogene). NEBNext® Ultra™ II DNA Library Prep Kit for Illumina E7645L