Strain Med15-myc was grown in rich medium (YES). a-myc was used for chromatin IP and DNA purified.
Extracted molecule
genomic DNA
Extraction protocol
For ChIP-on-chip experiments, cells were grown to mid-log phase in YES (5 g/l yeast extract, 30 g/l glucose and 0,1 g/l each of adenine, leucine, histidine, uracil, lysine, arginine and glutamine). 2*108 cells were fixed in 1 % formaldehyde for 30’, quenched in 125 mM glycine, harvested and washed twice in ice-cold PBS. Cells were resuspended in lysis buffer (150 mM NaCl, 0,1 % SDS, 1 % Triton X-100, 0,1 % Na-deoxycholate, 1 mM EDTA and 50 mM Hepes-KOH, pH 7,5) with protease inhibitors and lysed in the FastPrep machine (FP120, BIO101 Savant) with 1 volume of glass beads. The extracts were sonicated (Bioruptor UCD-200, Diagenode) to chromatin fragments of 600-1500 bp. Antibodies used for chromatin immunoprecipitation were anti-H3 (ab1791, Abcam) and anti-c-myc (M4439, Sigma). These were used with 30 ul of protein A sepharose slurry (17-5280-01, GE Healthcare) or 50 ul protein A beads (P-3391, Sigma). Crosslinks were reversed by overnight incubation at 65 degrees C, the immunoprecipitated DNA was treated with proteinase K and purified by phenol-chloroform extraction followed by ethanol precipitation. After RNAse A treatment, 2 ul of 40 ul was used as a template for realtime-PCR (Lightcycler 2.0, Roche). Enrichment was assessed against a negative control IP performed without antibody.
Label
Cy3
Label protocol
500 ng of amplified IP- and input DNA were labeled using Cy3-dCTP or Cy5-dCTP, and both samples were hybridized together to IGR + ORF arrays (Eurogentec) (Wiren et al., 2005). At least three independent ChIP-on-chip experiments were performed in each case, with dye-swap carried out in order to correct for dye bias.
Strain Med15-myc was grown in rich medium (YES). Total (=input) DNA was purified.
Extracted molecule
genomic DNA
Extraction protocol
For ChIP-on-chip experiments, cells were grown to mid-log phase in YES (5 g/l yeast extract, 30 g/l glucose and 0,1 g/l each of adenine, leucine, histidine, uracil, lysine, arginine and glutamine). 2*108 cells were fixed in 1 % formaldehyde for 30’, quenched in 125 mM glycine, harvested and washed twice in ice-cold PBS. Cells were resuspended in lysis buffer (150 mM NaCl, 0,1 % SDS, 1 % Triton X-100, 0,1 % Na-deoxycholate, 1 mM EDTA and 50 mM Hepes-KOH, pH 7,5) with protease inhibitors and lysed in the FastPrep machine (FP120, BIO101 Savant) with 1 volume of glass beads. The extracts were sonicated (Bioruptor UCD-200, Diagenode) to chromatin fragments of 600-1500 bp. Antibodies used for chromatin immunoprecipitation were anti-H3 (ab1791, Abcam) and anti-c-myc (M4439, Sigma). These were used with 30 ul of protein A sepharose slurry (17-5280-01, GE Healthcare) or 50 ul protein A beads (P-3391, Sigma). Crosslinks were reversed by overnight incubation at 65 degrees C, the immunoprecipitated DNA was treated with proteinase K and purified by phenol-chloroform extraction followed by ethanol precipitation. After RNAse A treatment, 2 ul of 40 ul was used as a template for realtime-PCR (Lightcycler 2.0, Roche). Enrichment was assessed against a negative control IP performed without antibody.
Label
Cy5
Label protocol
500 ng of amplified IP- and input DNA were labeled using Cy3-dCTP or Cy5-dCTP, and both samples were hybridized together to IGR + ORF arrays (Eurogentec) (Wiren et al., 2005). At least three independent ChIP-on-chip experiments were performed in each case, with dye-swap carried out in order to correct for dye bias.
Hybridization protocol
Both labeled DNA populations were hybridized onto the Eurogentec IGR+ORF arrays at 42°C overnight together with 10 ug of salmon sperm DNA.
Scan protocol
Arrays were scanned using a Versarray scanner and processed using Imagene.
Description
ChIP-DNA from a Med15-myc strain was hybridized on a Eurogentec array together with input DNA from the same preparation to evaluate genomic sites bound by Med15.
Data processing
Data analysis using Genespring GX software (v.7.3, Agilent Technologies) was carried out as follows: measurements less than 0.01 were set to 0,01, data was normalized to the 50th percentile and filtered on flags.
