Genechip hybridization wash and stain kit

RNA was isolated from human cell culture using Trizol-RNA lysis procedure (Thermo Fisher Scientific). RNA was DNaseI (Thermo Fisher Scientific) treated and then reversely transcribed by MMLV (Promega). Quantitative RT–PCR was performed in triplicate with SYBR select (Thermo Fisher Scientific) using Rotor-Gene 3000 (Qiagen) and normalized to GAPDH/ACTB level. Primers for RT–PCR are listed in Table S1.
We performed RNA microarrays (Clariom S human) according to manufacturer’s protocol (P/N 703174 Rev. 2) with 200 ng of total RNA. Reagents/equipment were GeneChip wt PLUS Reagent Kit, P/N: 902280; GeneChip Hybridization, Wash, and Stain Kit P/N 900720, GeneChip Scanner, GeneChip Fluidics Station 450, GeneChip Hybridization Oven 640, Bioanalyzer 2100 (Agilent), and RNA600 NanoKit (Agilent).

Total RNA was extracted using the TRIzol method. RNA concentrations were determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA). Total RNA from were screened for differentially expressed genes using an Agilent RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA), and a PrimeView Human GeneChip (Agilent, Santa Clara, CA, USA) was used for microarray analysis. RNA labelling and hybridisation to Agilent miRNA microarray chips were performed with a GeneChip Hybridization Wash and Stain Kit (Agilent, Santa Clara, CA, USA) (17 (link), 18 (link)). The GeneChip data was used to analyze by gene set enrichment analysis (GSEA), and differentially expressed genes were annotated using ingenuity pathway analysis (IPA) to predict path changes.