Yeast strains were grown in either standard rich glucose media (YPD) or minimal synthetic glucose (SD) media (supplemented only with uracil to compensate for auxotrophic deficiencies in the strains) to mid log phase (optical density of 1.0) at 30C in 500 ml culture flasks with agitation. Prior to harvesting, cells were fixed by the addition of formaldehyde to 1% final concentration in the media culture and agitated for 30 minutes, then quenched with the addition of 1M glycine to a final concentration of 0.1M. Cells were harvested by centrifugation and washed twice with Tris-Buffered Saline. Cells were stored at -80C until preparation.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin was prepared by resuspending the cells in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and bead beating with 0.7 mm zirconium beads for six rounds of 2 minutes, interspersed with cooling on ice. Cells were collected by centrifugation, the supernatant was discarded, and the pellet resuspended in lysis buffer. The lysate was sonicated for seven rounds of 30 seconds, 90% duty cycle, with a Misonix sonifier. Cellular debris was removed by centrifugation. For immunoprecipitation, anti-mouse magnetic Dynabeads (Invitrogen) were prepared by incubating with anti-cMyc (9E11) in phosphate-buffered saline containing 5 mg/ml BSA for several hours with rotation. Normalized amounts of protein was then incubated with the prepared beads in ChIP buffer (lysis buffer with 0.1% sodium deoxycholate). Immunoprecipitations were performed at 4C overnight with gentle rotation. Beads were harvested by magnetic field and washed twice with ChIP buffer, twice with ChIP buffer supplemented to 500 mM NaCl, twice with LiCl wash buffer (10 mM Tris pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with TE. Captured complexes were eluted and cross-links reversed by incubating the beads in buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS) at 65C overnight. DNA was purified using Qiagen PCR Clean-up columns using the manufacturer's protocol.
Label
Cy3
Label protocol
Purified ChIP eluate or Input DNA was denatured, incubated with a random primer (GTTTCCCAGTCACGATCNNNNNNNNN), and extended using Sequenase enzyme (US Biochemical) in the presence of dNTP. The reaction was denatured again and extended a second time with fresh Sequenase enzyme. A portion of the primed products was then amplified in a standard PCR reaction using the primer GTTTCCCAGTCACGATC and Platinum Taq (Invitrogen). Amplified products were purified by Qiagen PCR cleanup columns and verified by gel electrophoresis. A normalized amount (3 µg) was then labeled using Klenow enzyme, the primer GTTTCCCAGTCACGATC, 1 mM each of dATP, dTTP, dGTP, Cy3-dCTP or Cy5-dCTP, and 0.5 mM dCTP. Labeled products were purified using Qiagen PCR cleanup column with an additional 250 µl wash of 35% guanidine HCl.
Yeast strains were grown in either standard rich glucose media (YPD) or minimal synthetic glucose (SD) media (supplemented only with uracil to compensate for auxotrophic deficiencies in the strains) to mid log phase (optical density of 1.0) at 30C in 500 ml culture flasks with agitation. Prior to harvesting, cells were fixed by the addition of formaldehyde to 1% final concentration in the media culture and agitated for 30 minutes, then quenched with the addition of 1M glycine to a final concentration of 0.1M. Cells were harvested by centrifugation and washed twice with Tris-Buffered Saline. Cells were stored at -80C until preparation.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin was prepared by resuspending the cells in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and bead beating with 0.7 mm zirconium beads for six rounds of 2 minutes, interspersed with cooling on ice. Cells were collected by centrifugation, the supernatant was discarded, and the pellet resuspended in lysis buffer. The lysate was sonicated for seven rounds of 30 seconds, 90% duty cycle, with a Misonix sonifier. Cellular debris was removed by centrifugation. For immunoprecipitation, anti-mouse magnetic Dynabeads (Invitrogen) were prepared by incubating with anti-cMyc (9E11) in phosphate-buffered saline containing 5 mg/ml BSA for several hours with rotation. Normalized amounts of protein was then incubated with the prepared beads in ChIP buffer (lysis buffer with 0.1% sodium deoxycholate). Immunoprecipitations were performed at 4C overnight with gentle rotation. Beads were harvested by magnetic field and washed twice with ChIP buffer, twice with ChIP buffer supplemented to 500 mM NaCl, twice with LiCl wash buffer (10 mM Tris pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with TE. Captured complexes were eluted and cross-links reversed by incubating the beads in buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS) at 65C overnight. DNA was purified using Qiagen PCR Clean-up columns using the manufacturer's protocol.
Label
Cy5
Label protocol
Purified ChIP eluate or Input DNA was denatured, incubated with a random primer (GTTTCCCAGTCACGATCNNNNNNNNN), and extended using Sequenase enzyme (US Biochemical) in the presence of dNTP. The reaction was denatured again and extended a second time with fresh Sequenase enzyme. A portion of the primed products was then amplified in a standard PCR reaction using the primer GTTTCCCAGTCACGATC and Platinum Taq (Invitrogen). Amplified products were purified by Qiagen PCR cleanup columns and verified by gel electrophoresis. A normalized amount (3 µg) was then labeled using Klenow enzyme, the primer GTTTCCCAGTCACGATC, 1 mM each of dATP, dTTP, dGTP, Cy3-dCTP or Cy5-dCTP, and 0.5 mM dCTP. Labeled products were purified using Qiagen PCR cleanup column with an additional 250 µl wash of 35% guanidine HCl.
Hybridization protocol
Fluorescently labeled DNA samples were heat denatured after being combined with cot-1 DNA, Agilent aCGH blocking agent and Agilent Hi-RPM hybridization solution. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization Oven and were incubated at 65ºC for 24 hours with a rotational speed of 20 rpm. Following incubation, the microarray slide was washed for 5 minute in aCGH/ChIP-on-chip Wash Buffer 1 (0.5X SSPE, 0.005% N-lauroylsarcosine; room temperature) and 5 minute in aCGH/ChIP-on-chip Wash Buffer 2 (0.1X SSPE, 0.005% N-lauroylsarcosine; 31ºC). Microarray slides were briefly dipped in a solution of acetonitrile and dried.
Scan protocol
Microarray slides were scanned in an Agilent Technologies G2505C Microarray Scanner at 5 um resolution. The scanner performs simultaneous detection of Cyanine-3 and Cyanine-5 signal on the hybridized slide. Data captured from the scanned microarray image was saved as a TIF file. TIF files were loaded into Agilent Feature Extraction Software version 9.1.3.1 and feature intensities were calculated and exported as a text file.
Description
Ioc3 ChIP vs Input in YPD media, repeat 1 and 2 5289E3; 5290E3
Data processing
Raw feature intensities from two replicates were quantile normalized together, median scaled to 100, and the combined experiment values expressed as a log2 ratio of ChIP over Input. Probe sequences were mapped to Saccharoymyces cerevisisiae genome release 64 (UCSC sacCer3) and were then associated with the corresponding processed feature score. Processing was done with the BioToolBox (http://biotoolbox.googlecode.com) programs process_microarray and map_oligo_data2gff.
