TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation. Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound material was eluted from the beads by resuspending beads in 100 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation. Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30 µl of TE. These samples were further purified with Qiagen PCR product purification Kit.
Label
Cy5
Label protocol
IPDNA was labeled according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).
TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25 C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. 50 ul of WCE was mixed with 150 µl of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 50 µl of TE. These samples were further purified with Qiagen PCR product purification Kit.
Label
Cy3
Label protocol
WCE was labeled according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).
Hybridization protocol
IPDNA and WCE was hybridized according to Agilent recommended protocol Reference: Yeast ChIP on chip protocol, Agilent Technologies (version 9.1).
Scan protocol
Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.
Description
Microarray slides were obtained from Agilent Technologies (yeast 4 X 44k, whole genome array). The arrays comprise of 60 mer oligo nucleotides probes at ~ 290 bp spacing, covering ~12 Mb of the genome.
Data processing
Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol for array CGH that showed high correlation (r2=0.9) of bound probes between data of the two biological replicates. Identification of bound probes and genes was made using Agilent ChIP analytics software (version 1.3) by using Whitehead Institute Per-array Neighbourhood model v1.0. In the neighbourhood model, if candidate bound probe set p-value was less than 0.05, the central probe was marked as potentially bound, and the probe sets were required to pass one of two additional filters: the center probe in the probe set has a single probe p-value less than 0.05 or one of the flanking probes has a single point p-value less than 0.25. These two filters cover situations where a binding event occurs midway between two probes and each detects the event, weakly.
