To inhibit Ark1 kinase activity, 5µM of 1NM-PP1 were added 15 min before cells were released from G2 in minimal EMM2 medium at 25°C after a 6 h cdc25-22 imposed arrest at 36.5°C. The sample was taken 30 min after release when most cells were in anaphase.
Extracted molecule
genomic DNA
Extraction protocol
ChIP and microarray analysis were carried out essentially as previously described (Katou et al., 2003, see below). In short, 2x10^9 cells were fixed with 1% formaldehyde for half an hour at room temperature. Cell extracts were prepared using a multibeads shocker (Yasui Kikai). After sonication (Sanyo Soniprep150) genomic DNA fragments of 400-800bp were retrieved and taken for ChIP. We used anti-Pk mouse monoclonal SV5-Pk1 antibody (Serotec) against the Pk-tagged cohesin subunit Rad21 in conjunction with protein A magnetic dynabeads (Dynal). The eluted immunoprecipitates were incubated overnight at 65°C to reverse the cross-linking. The genomic DNA was purified and amplified by random primer PCR. Reference: Katou, Y., Kanoh, Y., Bando, M., Noguchi, H., Tanaka, H., Ashikari, T., Sugimoto, K. and Shirahige, K. (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature, 424, 1078-1083.
Label
biotin-11-ddATP (NEL548)
Label protocol
DNA was labeled with biotin-11-ddATP using terminal transferase TdT (Roche, 400U/µl).
Hybridization protocol
Labeled DNA was hybridised to microarrays in an Affymterix Gene Chip Hybridisation Oven 640 for 16h at 42°C & 60rpm. For washing and staining the EukGe_WS1_v4_450 fluidics protocol was run on an Affymetrix Gene Chip Fluidics Station (version 450). Staining included only one step with SAPE.
Scan protocol
Microarray scanning was performed with an Affymetrix Gene Chip Scanner 3000.
Description
no additional information
Data processing
The Affymetrix Gene Chip Operating Software GCOS, version 1.4, was used for data processing.
