7 protocols
Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:
cRNA from second T7 round amplification was labeled with Biotin-X-X-NHS for 1hr at 25C. (Parameters: Amplification = none, Mass unit = Micro gram)
normalization data transformation protocol
Normalized files corresponding to biological replicates were quantile normalized and averaged
normalization data transformation protocol
We used an adapted version of the Model-based Analysis of Tiling-arrays (MAT) algorithm to reliably detect enriched regions (Schulze, Gottardo, Kobor, manuscript in preparation) (Johnson et al., 2006). MAT was applied to corresponding immunoprecipitated and input sample array, and the probe behavior model was estimated by examining the signal intensity, sequence, and copy number of all probes on an array. After probe behavior model fitting, the residuals between the model and observation were normally distributed and centered at 0. MAT uses a score function to identify regions of ChIP enrichment, which allows robust p-value and false discovery rate calculations. MATscores are calculated from all probes within a 300-bp sliding window and returns a MATscore for each probe. All samples were normalized to input.
array scanning and feature extraction protocol
Data were scanned using MicroArraySuite 5.0 according to manufacturer specifications.
growth protocol
ChIP-on-chip cultures were grown overnight in YPD, diluted to 0.15 OD600 and grown to 0.5-0.6 OD600 units. Cultures were cross-linked with 1% formaldehyde for 20 min and harvested by centrifugation.
Chromatin immunoprecipitation and genome-wide location analyses were performed as described previously, using the adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (van Bakel et al., 2008). In brief, 500 ml yeast cells were grown in a rich medium to an OD600 0.8_0.9 and were cross-linked with 1% formaldehyde for 20 min before chromatin was extracted. The chromatin was sonicated (Bioruptor, Diagenode: 10 cycles,30s on/off, high setting) to yield an average DNA fragment of 500 bp. 4.2ul of anti-Flag (Sigma) antibody were coupled to 60 ul of protein A magnetic beads (Invitrogen). After reversal of the crosslinking and DNA purification, the immunoprecipitated and input DNA were amplified to about 6 ug aRNA using T7 RNA polymerase in two rounds. Samples were labeled with biotin, and the immunoprecipitated and input sample were hybridized to two Affymetrix 1.0R S. cerevisiae microarrays.