strain: BQS1217 strain (SAS3-HA tagged in wt strain BY4742)
Treatment protocol
Samples were crosslinked with 1% formaldehyde for 30 min, quenched by adding glycine to a final concentration of 125 mM and washed three times with 30 mL of ice-cold PBS buffer.
Growth protocol
Cells (40 mL of culture) were grow exponentially in rich medium (YPD) to OD600 0.5-0.8.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation was performed as previously described [Ren B, et al., 2000]. Briefly crosslinked cell pellets were resuspended in 300 μL of lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM PMSF, 1mM benzamidine and protease inhibitor cocktail from Roche) and lysed by vortexing at 4ºC by 45 min with glass beads. Chromatin was sonicated to 200–1000 bp (average of 400bp). Immunoprecipitation was carried for 4h using 2 µg of rat anti-HA 3F10 (Roche) Ab coupled to 100 µL of a 50% (v/v) suspension of Protein G Sepharose 4FF (Amersham Biosciences) equilibrated in 5mg/mL BSA lysis buffer. We included control immunoprecipitations of lysates from wild type strains with anti-HA. Beads were washed twice with lysis buffer, twice with 500 mM NaCl lysis buffer, twice with wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA pH 8.0, 1 mM PMSF, 1 mM benzamidine and protease inhibitor cocktail from Roche), once with TE containing 1mM PMSF, and finally collected. Two successive elutions were performed with elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubating 10 min at 65ºC. The eluted fraction was treated overnight at 65ºC to reverse the cross-linking. Proteins were degraded by proteinase K and DNA was purified by phenol/chloroform/isoamylic alcohol extraction. The sample as treated with RNAse A and purified. Ligation Mediated PCR was used for DNA amplification. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase, phenol/chloroform/isoamylic alcohol extracted and ethanol precipitated. Sample was ligated to annealed linkers oJW102 and oJW103. The sample was PCR amplified and amplified size analyzed on a 1.2% agarose gel (average size of 150 bp).
Label
Cy5
Label protocol
Labeling of DNA was obtained following the ‘Agilent Yeast ChIP-on-chip Analysis’ protocol Version 9.2 (Agilent Technologies, Palo Alto, California USA. p/n G4493-90010). 2000 ng of ChIP and reference samples were labeled respectively with Cyanine 5-dUTP and cyanine 3-dUTP using the ‘CGH Labeling Kit’ from Invitrogen (p/n 18095-011) according to the manufacturer’s instructions.
Samples were crosslinked with 1% formaldehyde for 30 min, quenched by adding glycine to a final concentration of 125 mM and washed three times with 30 mL of ice-cold PBS buffer.
Growth protocol
Cells (40 mL of culture) were grow exponentially in rich medium (YPD) to OD600 0.5-0.8.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin immunoprecipitation was performed as previously described [Ren B, et al., 2000]. Briefly crosslinked cell pellets were resuspended in 300 μL of lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM PMSF, 1mM benzamidine and protease inhibitor cocktail from Roche) and lysed by vortexing at 4ºC by 45 min with glass beads. Chromatin was sonicated to 200–1000 bp (average of 400bp). Immunoprecipitation was carried for 4h using 2 µg of rat anti-HA 3F10 (Roche) Ab coupled to 100 µL of a 50% (v/v) suspension of Protein G Sepharose 4FF (Amersham Biosciences) equilibrated in 5mg/mL BSA lysis buffer. We included control immunoprecipitations of lysates from wild type strains with anti-HA. Beads were washed twice with lysis buffer, twice with 500 mM NaCl lysis buffer, twice with wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA pH 8.0, 1 mM PMSF, 1 mM benzamidine and protease inhibitor cocktail from Roche), once with TE containing 1mM PMSF, and finally collected. Two successive elutions were performed with elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubating 10 min at 65ºC. The eluted fraction was treated overnight at 65ºC to reverse the cross-linking. Proteins were degraded by proteinase K and DNA was purified by phenol/chloroform/isoamylic alcohol extraction. The sample as treated with RNAse A and purified. Ligation Mediated PCR was used for DNA amplification. Immunoprecipitated DNA was blunted by T4 phage DNA polymerase, phenol/chloroform/isoamylic alcohol extracted and ethanol precipitated. Sample was ligated to annealed linkers oJW102 and oJW103. The sample was PCR amplified and amplified size analyzed on a 1.2% agarose gel (average size of 150 bp).
Label
Cy3
Label protocol
Labeling of DNA was obtained following the ‘Agilent Yeast ChIP-on-chip Analysis’ protocol Version 9.2 (Agilent Technologies, Palo Alto, California USA. p/n G4493-90010). 2000 ng of ChIP and reference samples were labeled respectively with Cyanine 5-dUTP and cyanine 3-dUTP using the ‘CGH Labeling Kit’ from Invitrogen (p/n 18095-011) according to the manufacturer’s instructions.
Hybridization protocol
Labeled DNA was hybridized to the Yeast Whole Genome ChIP-on-chip Microarray (Agilent p/n G4491A, AMADID: 014741) according to manufacture instruction.
Scan protocol
Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA) following the Agilent protocol ChIP_1010_Sep10, grid template 014741_D_F_20091202 and the metric set ChIP_QCMT_Sep10.
Description
exponential growth in YPD - Linear dye normalization
Data processing
Agilent Feature Extraction Software (v10.10.1.1) was used for background subtraction and linear normalization.
