Comment[GEAAccession]	E-GEAD-471
MAGE-TAB Version	1.1
Investigation Title	CENP-A ChIP-seq in WT and sfh1 mutant strains of Schizosaccharomyces pombe
Experiment Description	"We have previously shown that Sfh1, a core subunit of RSC, is involved in centromere function in fission yeast. To link the role of Sfh1/RSC in centromere function to chromatin regulation, we performed ChIP-seq of CENP-A(Cnp1) in WT and sfh1 temperature-sensitive mutant strains."
Experimental Design	strain or line design	binding site identification design	all pairs
Experimental Factor Name	immunoprecipitate	genotype
Experimental Factor Type	immunoprecipitate	genotype
Person Last Name	Tsunemine
Person First Name	Satoru
Person Affiliation	Laboratory of Bioorganic Chemistry, Department of Chemistry, Faculty of Sience
Person Roles	submitter
Public Release Date	2022-09-06
PubMed ID	36200823
Protocol Name	P-GEAD-1039	P-GEAD-1040	P-GEAD-1041	P-GEAD-1042	P-GEAD-1043
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	"S. pombe cells were cultivated to exponential growth phase in YES medium before temperatures were shifted to 36 degrees for 8 h to inactivate temperature-sensitive. 5 x 10^9 cells were fixed with 1% formaldehyde (Nacalai Tesque) for 20 min at 30 degrees C. After fixation was quenched with 150 mM glycine, cells were harvested and washed twice with Lysis Buffer (50 mM HEPES [pH 7.5], 1 mM EDTA, 1% TritonX-100, and 0.1% Na-deoxycholate: described in the previous section). The cell pellet was re-suspended in Lysis Buffer containing 1 mM PMSF and a protease inhibitor cocktail, and homogenized with a bead shocker (Yasui Kikai) 30 times for 60 sec each at 4 degrees C. Lysis buffer containing 1 mM PMSF, and a protease inhibitor cocktail was added to the resulting cell extracts to 2 mL. Cell extracts were sonicated for 240 sec using a New Bioruptor (CosmoBio) set at level \"\"\"\", centrifuged at 15,000 rpm for 15 min at 4 degrees C, and the resultant supernatant was used as the input fraction. The input fraction was subject to immunoprecipitation with secondary antibody-conjugated magnetic beads (Invitrogen) preincubated with antibody against the target protein or epitope. Beads were washed with Lysis Buffer and incubated for 2 h with cell extract at 4 degrees C. After immunoprecipitation, the beads were washed twice each with Lysis Buffer, Lysis/NaCl Buffer (50 mM HEPES [pH 7.5], 500 mM NaCl, 1 mM EDTA, 1% TritonX-100, and 0.1% Na-deoxycholate) containing 1 mM PMSF and protease inhibitor cocktail, Wash/LiCl Buffer (10 mM Tris [pH 8.0], 250 mM LiCl, 0.5% NP-40, and 0.5% Na-deoxycholate) containing 1 mM PMSF and protease inhibitor cocktail, and TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA)."	"Immunoprecipitated beads were then suspended in TE buffer containing RNase A and incubated for 1 h at 37 degrees C. Proteinase K (0.5 mg/mL) was added, and the mixture was incubated for an additional 1 h at 45?C. After reversal of crosslinks by incubation at 65 degrees C, DNA was purified by phenol/chloroform extraction, and qPCR was performed."	ChIP-seq samples were subsequently processed on the Illumina platform according to the manufacturer's instructions	Sequencing was performed using the manufacturer's standard procedure.	"Sequence reads were processed using BWA, SAM-tools and MACS. We calculated the IP to WCE ratio using wiggle format pileup files (step size: 10bp)."
SDRF File	E-GEAD-471.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB7615
Comment[Last Update Date]	2023-01-06