BMCRN_DN_TJ-Datafile10

Box plots of the signals from the microarray. The Affymetrix Clariom D Assay (Thermo Fisher Scientific), a next-generation microarray, was used for genome-wide transcriptome analysis. Labelled targets were prepared from RNA samples using the reagents and enzymes supplied in the GeneChip® WT Pico Reagent Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction with slight modifications. Total RNA (100 ng) from each sample was reverse-transcribed using T7 promoter-tagged oligo-dT primer and WT Pico First-Strand Enzyme. After the removal of excess primers, 3’ Adapter was added to the first-strand cDNA using WT Pico 3’ Adaptor Enzyme. The 3’ Adapter-tagged 1st strand cDNA was then subjected to polymerase chain reaction using WT Pico PCR Enzyme (Taq DNA polymerase) and adaptor-specific primers to synthesize T7 promoter-tagged double-stranded cDNA. The cDNA was subjected to in vitro transcription using WT Pico IVT Enzyme (T7 RNA polymerase) to synthesize complementary RNA. The complementary RNA was purified using Purification Beads supplied in the kit, and reverse-transcribed using random primers and WT Pico 2nd-Cycle ss-cDNA Enzyme to synthesize 2nd-cycle single-stranded cDNA. After hydrolysis of the template RNA using RNase H, the sense-strand cDNA (5.5 μg) was purified to remove RNase H, salts, and unincorporated dNTPs, then digested with uracil-DNA glycosidase into 40–70 nt fragments. Successful fragmentation was confirmed using an Agilent 2100 Bioanalyzer. The fragmented cDNA was then biotin-labeled with terminal deoxynucleotidyl transferase and subjected to hybridization according to the GeneChip® WT Pico Reagent Kit manual. The Clariom D array was processed through the automatic washing step using a GeneChip® Hybridization, Wash, and Stain Kit (Thermo Fisher Scientific) and Fluidics Station 450 (Thermo Fisher Scientific). Hybridised targets were stained with kit-provided streptavidin-phycoerythrin and detected using a Scanner 3000 7G (Thermo Fisher Scientific). Raw data (CEL files) were produced using Affymetrix GeneChip Command Console Software and processed using Affymetrix Expression Console Software. The CEL files were registered under Gene Expression Omnibus (GEO) accession no. GSE169393. A detection call algorithm was used to filter and remove missing expression values based on absent/present calls. Using this algorithm, present, marginal, or absent calls were obtained for each probe set in each array. A scaling factor was applied to the normalised data from the CEL files to bring the average intensity for all probes on the array to 500, generating CHP files for use in Microarray Suite 5. For gene expression comparisons, data assigned to absent calls were omitted