title: GSM8506074: untagged strain, Input; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: 100 mL of exponential culture (OD600 ~1) of untagged strain, WT CFP-cnp1 and SUMO-KallR CFP-cnp1 were mixed with 10 mL of budding yeast exponential culture Scc1-Pk, used as a calibrator and then crosslinked 1% formaldehyde (Sigma, 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 extracted by bead beating in FA-lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X100, 0.1% NaDeoxycholate, 1 mM EDTA with 1 mM PMSF and protease inhibitors (Sigma-Aldrich, P8215). Chromatin sonication was done in a DiagenodeBioruptor Pico using Easy mode 10 cycles of 30 s ON and 30 s OFF. Immunoprecipitation was carried o/n: 300 µL of extract was incubated on the wheel with anti-GFP antibody (Invitrogen, A11122) 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 decrosslinkedover night at 65° C. Next morning samples were digested with RNAse A (Thermo) for 30 min at 37oC and then with proteinase K at 65oC for 1h. The DNA associated with CFP-cnp1 was precipitated with isopropanol/glycogene/3M Kac for 1h in -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 ultra pure water (Sigma). DNA concentration was determined by qubit and test qPCR was ran on 10x dilution of obtained DNA suspension with SsoAdvanced Universal SYBR® Green Supermix (Biorad #1725274). NEBNext®Ultra™ II DNA Library Prep Kit for Illumina E7645L