Genechip human exon 1.0 st array

Total RNA was reverse-transcribed and hybridized onto Affymetrix HG-U133 Plus 2.0
array, using the identical protocol and ID names for genes as GeneChip Human Exon
1.0ST Arrays (Affymetrix, Santa Clara, CA, USA). The Affymetrix Human Gene Chip Exon
1.0 ST Array interrogates over one million exons representing over 17,868 NCBI
Reference Sequence (RefSeq) transcripts. Arrays were run using the
manufacturer's technical protocol (Affymetrix, Santa Clara, CA). Briefly, 2 qg
of total RNA was subjected to a ribosomal RNA removal procedure (RiboMinus Human/
Mouse Transcriptome Isolation Kit, Invitrogen - Thermo Fischer Scientific) to reduce
the 28S and 18S rRNA population to minimize background and increase sensitivity of
the assay. Reduced RNA was reverse-transcribed to cDNA using random hexamers tagged
with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA
polymerase (GeneChip WT cDNA Synthesis Kit, Affymetrix). The resulting
double-stranded cDNA was used for amplification of antisense cRNA and cleaned using
the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was
performed using random primers to reverse transcribe the cRNA into sense single
stranded DNA, which was fragmented, labeled, and hybridized to arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station and G7 Affymetrix
high-resolution scanner using GCOS 1.3.

After spiking total RNA from each cell line with bacterial poly-A RNA-positive controls (Affymetrix), every sample was reverse-transcribed, converted to double-stranded cDNA, in vitro-transcribed and amplified using the Ambion WT Expression Kit. The obtained single-stranded cDNA was biotinylated after fragmentation with the Affymetrix WT Terminal Labeling kit as outlined in the manufacturer’s instructions. The resulting samples were mixed with hybridization controls (Affymetrix) and hybridized on GeneChip Human Exon 1.0 ST Arrays (Affymetrix). The arrays were stained and washed in a GeneChip Fluidics Station 450 (Affymetrix) and scanned for raw probe signal intensities with the GeneChip Scanner 3000 (Affymetrix). For the processing of the data, see extended experimental procedures.