Genechip terminal labeling kit

Gene expression profiling was performed using the GeneChip^® Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) which interrogates 28,536 well-annotated genes with 253,002 distinct probe sets, allowing expression analysis at both gene and exon level. Ten µg of cRNA, synthesized and purified from 0.25 µg of total RNA using the Ambion^® WT Expression kit (Ambion, USA) was used for cDNA synthesis, followed by cDNA fragmentation and labeling with the GeneChip^® Terminal Labeling kit (Affymetrix). Fragmented and labeled cDNA was hybridized to Human Gene 1.0 ST arrays (Affymetrix) using the GeneChip^® Hybridization, Wash and Stain kit (Affymetrix) (hybridization module) and hybridization controls (Affymetrix) with rotation at 45°C for 16 hours. After hybridization, arrays were washed and stained using the GeneChip^® Hybridization, Wash and Stain kit (stain module) after which the arrays were immediately scanned using an Affymetrix GeneChip^® Scanner. Raw data of X-ray and heavy ion experiments have been submitted to the ArrayExpress database under accession numbers E-MTAB-3463 and E-MTAB-5761, respectively.

Using the Ambion^® WT Expression kit (Ambion, USA), cDNA was prepared from 10 μg of purified cRNA, originally synthesized and purified from 0.25 μg of total RNA, following the manufacturer’s instructions. The cDNA (2.75 μg) was used for fragmentation and labeling using GeneChip^® Terminal Labeling kit (Affymetrix, USA). Using GeneChip^® Hybridization, Wash and Stain kit (hybridization module) (Affymetrix), and hybridization controls (Affymetrix), fragmented and labeled cDNA was hybridized to Human Gene 1.0 ST arrays (Affymetrix). After hybridization with rotation for 16 h at 45°C, arrays were washed and stained, according to the manufacturer’s instructions, using GeneChip^® Hybridization, Wash and Stain kit (stain module) (Affymetrix). Finally, arrays were scanned immediately using Affymetrix GeneChip^® Scanner.