Strain: delta-hrp1 Grown in rich medium (YES) ChIP performed using anti-H3 antibody
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
Cy 3
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: delta-hrp1 Grown in rich medium (YES) Input DNA
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
Cy 5
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 delta-hrp1 strain was hybridized on a Eurogentec array together with input DNA from the same preparation to evaluate the histone 3 density in the strain.
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.
