Genechip s pombe tiling 1.0fr

Genomic DNA was extracted as described previously [7 (link)], with an additional clean-up of genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen). DamID experiments were done as described by [54 (link)]. The amplified material was cleaned using the Qiagen PCR purification kit and fragmented with DNAse. Fragmented DNA was labeled and hybridized to the Affymetrix GeneChip S. pombe Tiling 1.0FR using standard protocols by the Affymetrix core facility at Novum (http://apt.bea.ki.se).
All experiments were done as biological duplicates.

For microarray hybridization, immunoprecipitated DNA was amplified as described by Durand-Dubief et al [22 (link)]. Fragmentation, labeling and hybridization to the Affymetrix GeneChip S. pombe Tiling 1.0FR were performed by the BEA core facility at Novum (http://www.bea.ki.se) according to Affymetrix standard protocols. For domainogram visualization, raw data from Affymetrix (.CEL format) were normalized using Affymetrix Tiling Analysis Software (TAS) v1.1. Duplicate data were normalized to input using quantile normalization plus scaling using a bandwidth of 100. Data obtained from TAS were then directly loaded into the Seqmonk program, and the antibody background was removed using data for the clr4∆ mutant. Data were visualized using the following parameters: number of classes, 30; min probe count, 30; and max probe count, 100. Raw and normalized ChIP microarray data have been submitted to the Gene Expression Omnibus under accession number GSE116038.

DNA was immunoprecipitated as described earlier [53 (link)] using 2μl of anti-H4K12Ac (ab1761, abcam), 2μl of anti-myc (9E10, Sigma), 1 μl of anti-GFP (ab290, abcam), or 3μl anti-RNA polymerase II CTD repeat (ab5408, abcam) antibodies per 100μl chromatin extracts.
For microarray hybridization, immunoprecipitated DNA was amplified to 5 μg DNA as described in [53 (link)], with the exception that in the second PCR, 5 mM dUTP was added to the reaction. Fragmentation, labeling and hybridization to the Affymetrix GeneChip S. pombe Tiling 1.0FR was performed by the Affymetrix core facility at Novum (http://apt.bea.ki.se) according to Affymetrix standard protocols.
All experiments were done as biological duplicates.
For real-time quantitative PCR, immunoprecipitated DNA was amplified in the presence of SYBR Green using Applied Biosystems 7500 real-time PCR machine. The primers used are listed in S2 Table.