Cells were treated with 2 or 5 uM of inhibitor (1-NAPP-1 or CMK) or equivalent solvent control (DMSO) at mid-log phase (50 mL, OD600 0.4 to 1) for 1 hr before crosslinking with 1% formaldehyde for 5 min.
Growth protocol
Yeast were grown in His-dropout media + 2% dextrose at 30 °C in a shaking incubator from an OD600 of 0.1 to mid-log phase OD600 of 0.5 to 1.
Extracted molecule
genomic DNA
Extraction protocol
For every immunoprecipitation, 50 ul of protein G conjugated Dynabeads (Life Technologies), pre-washed with lysis buffer (50mM HEPES-KOH [pH 7.5], 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 1mM, Aprotinin, 1mM Leupeptin, 1mM PMSF, 1mM Benzamidine, 1.45mM Pepstatin, 1mM NaN3, 1mM Na3VO4, 1mM NaF), were mixed with the whole cell extract-antibody mixture to pulldown Pol II complexes. Washes and elution were performed as previously described (Tietjen et al., 2010).
Label
Cy5
Label protocol
ChIP DNA was end-repaired, linkers were ligated and ligation-mediated PCR was performed as previously described (Chinchilla et al., 2012).
Channel 2
Source name
Input DNA from kin28is budding yeast treated with CMK
For every immunoprecipitation, 50 ul of protein G conjugated Dynabeads (Life Technologies), pre-washed with lysis buffer (50mM HEPES-KOH [pH 7.5], 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 1mM, Aprotinin, 1mM Leupeptin, 1mM PMSF, 1mM Benzamidine, 1.45mM Pepstatin, 1mM NaN3, 1mM Na3VO4, 1mM NaF), were mixed with the whole cell extract-antibody mixture to pulldown Pol II complexes. Washes and elution were performed as previously described (Tietjen et al., 2010).
Label
Cy3
Label protocol
ChIP DNA was end-repaired, linkers were ligated and ligation-mediated PCR was performed as previously described (Chinchilla et al., 2012).
Hybridization protocol
24 pmol of ChIP DNA and an equivalent mass of input DNA were mixed in a speedvac on medium heat for 30 min. The samples were resuspended in 18 uL of hybrdization buffer and loaded using NimbleGen X1 mixers and hybridzed on a MAUI instrument.
Scan protocol
Arrays were scanned on a GenePix 4000B instrument as previously described (Chinchilla et al., 2012).
Data processing
Probe intensities were extracted with NimbleScan using the .ndf file (attached). Global and local normalization was performed using in-house R code available upon request, log2-transformed and averaged with a five-probe moving average to smooth the data.
Pol II and CTD Phosphorylation of ATP-analog sensitive Kin28 budding yeast
Data table header descriptions
ID_REF
VALUE
ChIP-chip immunoprecipitated data was mean-scaled against its respective 'input' sample data, and then the ratio of scaled immunoprecipitation to the input was log2-transformed
