Genechip wt terminal labelling kit

Total RNA was converted to cRNA using the WT Expression kit (Ambion). Quality of cRNA was assessed with a 2100 Bioanalyser. cRNA was converted to second-strand cDNA using the WT Expression kit (Ambion). cDNA was fragmented and labelled using the GeneChip WT Terminal Labelling kit (Affymetrix). Labelled cDNA was hybridised to a GeneChip Mouse Gene 1.0 ST Array. GeneSpring software was used for data analysis and quality control. Probes were normalised by quantile normalisation among all microarray data.

Microarray data were collected at Expression Analysis, Inc. (www.expressionanalysis.com; Durham, NC). Biotin-labeled target for the microarray experiment was prepared using 100 ng of total RNA and cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix, Santa Clara, CA). The sense cDNA was then fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidic endonuclease 1) and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labelling kit (Affymetrix). Hybridization was performed using 5 micrograms of biotinylated target, which was incubated with the GeneChip Human Gene 1.0 ST array (Affymetrix) at 45°C for 16–20 hours. Each sample was hybridized with a dedicated array. Following hybridization, non-specifically bound material was removed by washing and detection of specifically bound target was performed using the GeneChip Hybridization, Wash and Stain kit, and the GeneChip Fluidics Station 450 (Affymetrix). The arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix) and raw data were extracted from the scanned images and analyzed with the Affymetrix GeneChip Command Console Software (AGCC, Affymetrix).
Microarray data have been deposited in the NCBI Gene Expression Omnibus (Accession Number GSE58682).

A sense-strand cDNA generated from the total RNA using an Ambion WT Expression Kit (Thermo Fisher Scientific, Waltham, MA, USA) was fragmented and labelled using the GeneChip WT Terminal Labelling Kit (Affymetrix, Santa Clara, CA, USA) and next hybridized onto an Affymetrix Human Gene 2.1 ST Array Strip. The hybridization and subsequent fluidics and scanning steps were carried out with an Affymetrix GeneAtlas System, with designated software.

Experiments were designed with three independent biological replicas. Biotinylated cDNA targets were prepared, starting from 150 ng of total RNA, using the Ambion WT Expression Kit (Cat 4411974), and the Affymetrix GeneChip WT Terminal Labelling Kit (Cat 900671) according to Affymetrix recommendations. Following fragmentation and end-labeling, 3 μg of cDNAs were hybridized on GeneChip Mouse Gene 2.0 ST arrays (Affymetrix, UK) for whole-transcript expression profiles. Washed and stained chips were scanned with the GeneChip Scanner 3000 7 G (Affymetrix) at a resolution of 0.7 μm. Obtained raw data (.CEL intensity files) were processed with Affymetrix Expression Console software version 1.1 to calculate probe set signal intensities using Robust Multi-array Average (RMA) algorithms with default settings.
To select the DE genes, we used the fold change rank ordering statistics (FCROS) method (Dembele and Kastner, 2014 (link)). In the FCROS method, k pairs of test/control samples are used to compute fold changes (FC). For each pair of test/control samples, obtained FCs for all genes are ranked in increasing order. Ranks that result are associated to genes. Then, the k-ranks of each gene are used to calculate a statistic, and resulting probability (f-value) is used to identify the DE genes with an error level of 5%.

RNA labelling and hybridization were carried out by Edinburgh Genomics, University of Edinburgh (https://genomics.ed.ac.uk/ Accessed 13 July 2021). For microarray, cDNA was produced using the Ambion WT expression kit (Invitrogen) and accordingly labelled using the GeneChip WT terminal labelling kit (Affymetrix).
Approximately 3 µg of fragmented, biotin-labelled cDNA was hybridised to a Mouse Gene 2.1 ST array plate (Affymetrix) using the Gene Titan instrument (Affymetrix) and standard Affymetrix protocols.