ChIP-chip Analysis of Transcription Factors

The ChIP-chip experiments were performed as described previously [19 (link)]. Briefly, Cmr1 and Rpb3 ChIP DNA and corresponding input DNA from WT, cmr1Δ or gcn4Δ were amplified using the GenomePlex complete whole-genome amplification kit (Sigma, WGA2) as per manufacturer instructions. The ChIP and input DNA were labeled by Alexa555 and Alexa647, respectively using BioPrime Plus Array GCH Labelling System (Invitrogen, 18095–013). Equal quantity of labeled ChIP and input DNA were hybridized and processed as per the manufacturer instructions. The arrays were scanned on G2505C Agilent SureScan microarray scanner and data was extracted using Agilent Feature Extraction software.
The feature-extracted data was read into R software and normalized using Limma package from Bioconductor as described previously [19 (link)]. The ORF occupancies of Cmr1 and Rpb3 were determined by averaging normalized ChIP/input log₂ values for the probes present within the transcription start site (TSS) and the transcription end sites (TES). Gene-average profiles were generated using the versatile aggregate profiler [22 (link)] as described previously [23 (link)].