Affymetrix genechips

At the end of high temperature stress, fresh flag leaves were used for gene transcriptome analysis with the Affymetrix GeneChips (Affymetrix, Santa Clara, CA, USA). The analysis was conducted in the Capitalbio Corporation in Beijing according to the instructions for Affymetrix (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). GeneChip Operating Software 1.4 was used to analyze the hybridization data under the criteria of at least 2.0-fold changes (log₂ values), with a false discovery rate <0.05 at gene expression level. Functional annotation was performed based on Arabidopsis thaliana genome databases.

Total RNA was extracted from NCI-H929 cells 24 h post-treatment with the corresponding IC₅₀ value of crizotinib or with DMSO as a negative control using the RNeasy Kit from Qiagen (Hilden, Germany). Duplicate samples were subjected to microarray hybridization for gene expression profiling using Affymetrix GeneChips^® (Affymetrix, Santa Clara, CA, USA) with human Clariom S assays as described previously [55 (link),56 (link),57 (link)] at the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ, Heidelberg, Germany).

For the analysis on Affymetrix GeneChips (Affymetrix, Santa Clara, CA) 50 ng of immunoprecipitated RNA or of a total RNA control sample were processed with the two-cycle target labelling protocol (Technical Manual; Gene Expression Analysis 701021 Rv.3 of Affymetrix) to produce biotinylated cRNA target. 11 μg of this amplified material of each sample were fragmented and hybridized to GeneChip Mouse 430 2.0 arrays. Washing and staining was performed using the EuGE-WS2v5 protocol. Scanning was done on the Affymetrix GeneChip Scanner 7G. Expression signals were normalized by robust multiarray average (RMA) [40 ]. Differentially hybridizing features were identified using Limma [41 ] and adjusted for multiple testing with the Benjamini and Hochberg’s method. Alternatively, Z scores and Z ratios were calculated according to [42 (link)]. Microarray data are available at NCBI Gene Expression Omnibus at accession number GSE64761.