strain: Y0003 chip antibody: IgG (unspecific rabbit IgG) raw data: 532303A04_532.pair raw data: 532303A04_635.pair
Growth protocol
Yeast cell precultures were grown over-night in YP+2% glucose medium and main cultures were harvested in mid-log-phase (OD600 0.7-1). All cultures were grown under constant shaking at 30°C, except for pre-cultures for temperature sensitive strains (cdc48-6, cdc48-3, ufd1-2) grown at 25°C.
Extracted molecule
genomic DNA
Extraction protocol
ChIP was performed based on a protocol (Aparicio et al, 2005), with minor modifications as described (Kalocsay et al, 2009). Briefly, for each IP yeast cells equivalent to 75 OD600-units were crosslinked with formaldehyde (1% final) for 16 minutes and the reaction was quenched by adding glycine (325mM final). Cells were lysed by beat-beating (MM301, Retsch GmbH) with zirconia/silica beads (BioSpec Inc.), chromatin was isolated and sheared to 200-500 bp fragments using a Bioruptor UCD-200 sonication system (Diagenode). Cell debris was pelleted by centrifugation, input samples were collected and chromatin was subjected to immunoprecipitation with Protein A Sepharose CL-4B (GE Healthcare) and specific antibodies as follows: ubiquitin (clone FK2, Merck/Millipore), ubiquitin-K48-specific (Apu2, Merck/Millipore), Myc-tag (9E10, Sigma), unspecific rabbit IgG (Bethyl Laboratories Inc.) and specific rabbit antibodies raised against Ymr111c/Euc1 (aa 292-462). Enriched DNA was de-crosslinked and subjected to Proteinase K digest (Sigma), purified (Qiagen PCR purification kit) and processed for genome-wide quantification using NimbleGen arrays. ChIP-chip was performed as described before (Kalocsay et al, 2009; Renkawitz et al, 2013). Briefly, input and ChIP DNA samples were treated with RNase (Sigma) and amplified with the GenomePlex Complete Whole Genome Amplification kit (Sigma) in two steps as described (O'Geen et al, 2006). Labeling of input and ChIP DNA (Cy3 or Cy5), hybridization to S. cerevisiae tiling arrays, array scanning and raw data extraction was performed by Source BioScience Berlin (formerly imaGenes, NimbleGen ChIP-chip service). Custom-designed c12plex NimbleGen arrays with 84 bp median genomic probe spacing and only unique probes were used. A dye-swap was included for replicate experiments and genome-wide binding profiles were generated from two independent experiments (except for ub-FK2 rad6-delta-and IgG WT profiles in Fig. 1A, performed once).
Label
Cy3 (input),Cy5 (ChIP)
Label protocol
standard NimbleGen protocol
Hybridization protocol
standard NimbleGen protocol
Scan protocol
standard NimbleGen protocol
Data processing
Raw signals of all channels in all replicate arrays were normalized and log2 transformed using the 'vsn' package in R/Bioconductor as described ((Prestel et al., 2010) and Tobias Straub Epigenome project PROT43, http://www.epigenesys.eu/).
