Comment[GEAAccession]	E-GEAD-427
MAGE-TAB Version	1.1
Investigation Title	The exploration of cooperative bindings of two transcription factors by ChIP-seq of Atf1-Rst2 binding during glucose starvation in Schizosaccharomyces pombe.
Experiment Description	Transcriptional regulation is pivotal biological process to adapt to environmental fluctuations and is achieved by the binding of numerous transcriptional factors (TFs) to their target sequences. TFs determine which genes to activate through binding to their binding sequences consisting of specific motif. However, how TFs choose appropriate targets from the numerous candidates in huge genome has not been fully elucidated. We found that the bindings of TFs to the binding sequences placed in close proximity cooperatively stabilize their binding, and thereby integrating distinct signal-pathways in fission yeast fbp1 gene. This study aims to identify cooperative two TFs binding sites. To this end, we co-precipitated genome DNA to where both TFs, Atf1 and Rst2, bind by the sequential-ChIP analysis using anti-Flag and anti-Atf1 antibodies in rst2-3flag cells, and analyzed the resultant ChIP DNA samples with next generation sequencer.
Experimental Design	cellular modification design	stimulus or stress design
Experimental Factor Name	glucose
Experimental Factor Type	glucose
Person Last Name	Senmatsu	Hirota
Person First Name	Satoshi	Kouji
Person Affiliation	Hirota Lab, Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University	
Person Roles	submitter	submitter
Public Release Date	2025-04-17
Protocol Name	P-GEAD-809	P-GEAD-810	P-GEAD-811	P-GEAD-812	P-GEAD-813
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	Fifty ml of culture was incubated with 37% formaldehyde (1.4 ml) solution for 20 min at room temperature, after which 2.5 M glycine (2.5 ml) was added and incubated for 5 min. After centrifugation, collected cells were washed twice with ice-cold TBS buffer (150 mM NaCl, 20mM Tris HCl [pH 7.5]).	The cells were mixed with lysis 140 buffer (400 ul) (0.1% Na-deoxycholate, 1 mM EDTA, 50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, and 1% Triton X100) and zirconia beads (0.6 ml) were added. After disruption of cells using a multi-beads shocker (Yasuikikai, Osaka, Japan), the suspension was sonicated 6 times for 30 sec each to shear chromosomal DNA into fragments (about 500 bp) and centrifuged at 4 degree C, after which the supernatant was collected as a whole-cell extract. The recommended amount of antibodies (anti-Atf1 [abcam ab18123] and Anti-DYKDDDDK [Wako 018-22383]) according to the specifications provided by the manufacturer, and DYNA-protein A beads (20 ul) (DYNAL, Oslo, Norway) were mixed at 4 degree C overnight to conjugate antibodies and beads. Whole-cell extract (300 ul) was mixed with pretreated Anti-DYKDDDDK-beads complex and allowed to immunoprecipitate overnight at 4 degree C. The precipitates were washed twice with lysis 140 buffer and once with lysis 500 (0.1% Na-deoxycholate, 1 mM EDTA, 50 mM HEPES-KOH [pH 7.5], 500 mM NaCl, and 1% Triton X100), then washed a further two times with wash buffer (0.5% Na-deoxycholate, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 10 mM Tris-HCl [pH 8.0]), then washed a final time with TE (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). The well-washed precipitates were mixed with TBS buffer (100 ul) containing 5 mg/ml of 3-FLAG peptide (Sigma) and held at room temperature for 30 min to allow for the elution of co-precipitant. Eluted materials were diluted by lysis 140 buffer (300 ul) and mixed with pretreated Anti-Atf1-beads complex and allowed to immunoprecipitate overnight at 4 degree C. The precipitates were washed as described above and allowed to elute the immunoprecipitated protein-DNA complexes in elution buffer (40 ul) (10 mM EDTA 1% SDS and 50 mM Tris-HCl [pH 8.0]) at 65 degree C for 10 min. The samples were then mixed with TE buffer (250 ul) containing 1% SDS and 63 ug of proteinase K (Merck, Darmstadt, Germany) and incubated at 37 degree C for 16 h. After incubation, the temperature was shifted to 65 degree C and the samples were incubated for a further 6 h. After incubation, DNA was phenol/chloroform extracted from each of the samples.	NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB, MA) with the END repair/dA-tailing module and Index primers (NEB, MA)	HiSeq X Ten paired end sequencing	ChIP control samples (ChIP without antibodies)
SDRF File	E-GEAD-427.sdrf.txt
Comment[AEExperimentType]	ChIP-seq
Comment[BioProject]	PRJDB11452
Comment[Last Update Date]	2025-04-17