RNA was collected into RNALater and RNA extracted with Trizol reagent, followed by column extraction with AmbionPure Link kit in accordance with the manufacturers protocol and eluting in 50μl. RNA was quantified using the nanodrop 2000 (thermo Scientific).
Label
Biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
1
Data processing
First the data was re-annotated using illuminaHumanv4.db bioconductor R package. This included exclusion of “bad” probes and assignment of probes to correct genes (see Barbosa-Morais et al. (2010) A re-annotation pipeline for Illumina BeadArrays: improving the interpretation of gene expression data. Nucleic Acids Research). The reannotated data were analysed using RUV-inv (Gagnon-Bartsch JA, Jacob L, Speed TP. Removing unwanted variation from high dimensional data with negative controls. Statistics Technical Reports, Department of Statistics, University of California, Berkley, USA. 2013; Gagnon-Bartsch JA, Speed TP. Using control genes to correct for unwanted variation in microarray data. Biostatistics. 2012;13(3):539-552). This method does not include a traditional normalisation step, and therefore only matrices of original and reannotated, but still non-normalised data are provided.
