RNA was extracted using the Qiagen RNeasy mini kit (Qiagen, Crawley, UK).
Label
Biotin
Label protocol
Total RNA was converted to labelled cDNA for hybridization onto the gene expression chips using the AmbionTM WT Expression Kit. The single-stranded DNA was fragmented and labelled using Affymetrix GeneChipTM WT Terminal Labelling Kit *.
Hybridization protocol
Hybridization was performed for 16 h on GeneTitanTM Instrument.
Scan protocol
GeneChips were scanned on a GeneTitan system (Affymetrix Ltd.).
Data processing
The Robust Multichip Analysis (RMA) algorithm, as implemented in Affymetrix Power Tools (APT, version 1.15.0), was utilized for the generation of normalized transcript cluster signal values. Systematic variation in expression between time points and conditions was modeled using LIMMA. Three contrast matrices were generated, first extracting contrasts between time-points under control conditions, second extracting contrasts between time-points under knockdown conditions, and third identifying transcript clusters that respond differently over time between the two conditions. P-values were adjusted using Benjamini and Hochberg's method in order to control the false discovery rate (FDR). Universally low expressed transcript clusters (i.e. those expressed below the median across all 48 arrays) were excluded.
