Genechip human mapping 250k nsp assay kit

For expression analysis, total RNA was isolated from 10⁷ cells using the RNeasy Mini Kit (Qiagen) and cDNAs were generated from 25 ng of total RNA and subjected to PCR amplification (17 cycles) using the TransPlex^® Complete Whole Transcriptome Amplification Kit (Sigma, WTA2) according to manufacturer's instructions. 8 μg of amplified cDNAs were fragmented and labeled using the GeneChip Human Mapping 250K Nsp Assay kit (Affymetrix, 900766) according to manufacturer's instructions. Labeled cDNAs were then hybridized in GeneChip Drosophila Genome arrays 2.0 from Affymetrix (Thermo Fisher, 900531) for 16 h at 45°C. After incubation, the arrays were processed in the GeneChip Fluidics Station 450 from Affymetrix and scanned in GSC3000 System from Affymetrix.
For RT-qPCR analysis, total RNA was isolated from 10⁷ cells using the RNeasy Mini Kit (Qiagen). For cDNA synthesis, 1 μg of total RNA was used and qPCRs were run in triplicate in at least 4 independent experiments. Expression data were normalized to Rpl32 and analyzed using ΔΔCt method. Primers used in these experiments are described in Supplementary Table S1.