title: GSM2884444: S. cerevisiae cSpp1_GFP_t360_rep1 competition ChIP experiment; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: 50 mL of meiotic cells (4x10^7 cells/mL) were collected at the indicated time points and crosslinked with 1% formaldehyde for 20 min at room temperature. Formaldehyde was quenched with 125 mM glycine for 5 min at room temperature, and cells were washed three times with ice-cold 1x TBS at pH 7.5 (20 mM Tris HCL at pH 7.5, 150 mM NaCl). Cells were resuspended in 500  µL of lysis buffer (50 mM Hepes KOH at pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 tablet of complete inhibitor cocktail (from Roche) in 50 mL solution) and lysed with acid-washed glass beads for 10 min in a FastPrep bead beater machine. Chromatin samples was fragmented to an average size of 300 bp by sonication (Bioruptor, Diagenode). To obtain whole-cell extract (WCE), a 50-µL pre-IP sample was removed and centrifuged at full speed for 10 sec to pellet the cell debris (supernatant = WCE). The rest of the samples were also centrifuged at 12,000 rpm (4 °C) for 20 sec to pellet the cell debris. IP was performed by adding the 450-µL extract to a pellet of magnetic protein G dynabeads (Dynal), corresponding to 50 µL or 2 x 10^7 beads, which were preincubated with the 9E11 (monoclonal mouse anti-myc, ab290, Abcam) or anti-GFP (polyclonal rabbit, ab56, Abcam) antibodies overnight at 4 °C. IP samples were washed twice with lysis buffer, twice with lysis buffer plus 360 mM NaCl, twice with washing buffer (10 mM Tris HCl at pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA), and finally once with 1x TE at pH 7.5, using the magnetic device supplied by Dynal. After reversal of cross-linking by heating in TE-1% SDS overnight at 65 °C, the proteins were digested with proteinase K (12 µL of 20mg/mLstock) for 3h at 65 °C. Nucleic acids were PCR clean up kit purified and RNA digestion (10 µg RNase) was carried out for 1 h at 37°C. The DNA was finally resuspended in 50 µL nucl Sequencing libraries were prepared according to the Illuminas TruSeq ChIP Sample Preparation protocol. Briefly, the enriched ChIP DNA was end-repaired and indexed adapters were ligated to the inserts. Purified ligation products were then amplified by PCR. Amplified libraries were prepared in the Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen, Hungary. The libraries were sequenced using 50 single end reads with Illumina HiScan SQ (Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen); or with Illumina HiSeq 2500 (EMBL Genomics Core Facility, Heidelberg, Germany
attributes.tag: GEO Accession
attributes.value: GSM2884444