Genechip terminal labelling kit

Total RNA was extracted from flash frozen cells using the AllPrep DNA/RNA/protein Mini kit (Qiagen, Hilden, Germany), quality-controlled using a 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA) and quantified using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, USA). Only samples with an RNA Integrity Number ‘RIN’ >8.0 were used for further gene expression analysis. Using the WT Expression Kit (Ambion Inc, Austin, TX, USA), cDNA was prepared from 10 μg of purified cRNA, originally synthesised and purified from 0.25 μg of total RNA following the manufacturer's instructions. The cDNA (2.75 μg) was then used for fragmentation and labeling using GeneChip Terminal Labelling Kit (Affymetrix, Santa Clara, CA, USA). Using GeneChip Hybridization, Wash and Stain (Hybridization module) (Affymetrix, Santa Clara, CA, USA), and Hybridization controls (Affymetrix, Santa Clara, CA, USA), fragmented and labelled cDNA was hybridized to Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA, USA). After hybridization under orbital rotation for 16 h at 45°C, arrays were washed and stained using GeneChip Hybridization, Wash and Stain Kit (Stain module) (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions. Finally, arrays were scanned immediately using Affymetrix GeneChip Scanner GS 3000.

The total RNA was extracted using the mirVANA miRNA isolation kit (Ambion) according to the manufacturer’s instructions, and its integrity was assessed using a Bioanalyzer (Agilent). The samples were prepared using the WT Expression Kit (Ambion) and the GeneChip Terminal Labelling Kit (Affymetrix) according to the manufacturer’s instructions, and were hybridised to the Mouse Exon 1.0 ST Array (Affymetrix). The data was analysed using GeneSpring GX (Agilent). The detailed method used for quantitative RT-PCR has been previously described elsewhere^{39 (link)}. Briefly, it was performed using the SYBR green mix (ABI) and gene-specific primers using the Thermal Cycler Dice Real Time System II (Takara Bio). More than three biological replicates per group and three technical replicates were applied. The relative mRNA expression was calculated using the standard curve method by normalizing the absolute expression level to that of ß-actin and relative to that of the control samples. The sequences of primers designed using the Primer3 software are available in Supplementary Table S2.