Clariom d array

For transcriptomics analysis, we used Affymetrix Clariom D array for humans, containing over 6 million probes for protein-coding genes but also protein non-coding genes such as miRNAs, lncRNAs, and small nucleolar RNAs (snoRNAs) (Thermo Fisher Scientific, Santa Clara, CA). RNA (100 ng) was used to prepare cRNA and sscDNA using Thermo Fisher Scientific GeneChip^® WT PLUS reagent Kit. SscDNA, in an amount of 5.5 μg, was fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) and labeled by terminal deoxynucleotidyl transferase (TdT) using the DNA Labeling Reagent that is covalently linked to biotin. Fragmented and labeled ssCDNA samples, in triplicate, were used for hybridization, staining, and scanning by using Thermo Fisher Scientific WT array hybridization protocol following the manufacturer’s protocol, at the UC Davis Genome Center shared resource core. Hybridization of fragmented and labeled ssCDNA samples was done using GeneChip^TMHybridization oven and the arrays were washed then stained using GeneChip^TM Fluidics Station. The arrays were scanned using GeneChip^TM Scanner 3000 7G (Thermo Fisher Scientific, Santa Clara, CA). Quality control of the microarrays and data analysis were performed using Thermo Fisher Scientific Transcriptome Analysis Console software version 4.0.2.

Data generation was performed according to the vendor’s original protocol using GeneChip™ WT Pico Reagent Kit (Thermo Scientific, USA), followed by hybridization with the Clariom D array (Clariom™ D Pico Assay, human, Thermo Scientific). Clariom D array data were imported into R (v4.1.2) using the oligo package (v1.60.0) preprocessed utilizing the robust multichip average algorithm (RMA). Annotations were added using the annotateEset function implemented in the affycoretools (v1.68.0) package and clariomdhumantranscriptcluster.db (v8.8.0) as annotations. Probes not matching known genes were removed before analysis.

Single-stranded cDNA library was generated from DNase treated 1 μg of total RNA (3 WT vs. 3 Atxn2-CAG100-KIN cerebella), using GeneChipTM WT PLUS Reagent Kit (Applied Biosystems, Thermo Scientific, Waltham, MA, USA). Fragmentation and labeling of the cDNA library was performed immediately before hybridization to a Clariom D Array (Thermo Scientific, Waltham, MA, USA). The microarrays were scanned with the Affymetrix GeneChip Scanner, and data analysis was done with the Transcriptome Analysis Console (TAC) 4.0.1 (Applied Biosystems, Thermo Scientific, Waltham, MA, USA) using default parameters. For STRING interaction and pathway enrichment analysis, default parameters were employed.

The aortic intima-medial layer was separated manually from the adventitia, and mRNA was extracted from the intima-medial using RNeasy Mini kit (Qiagen, Crawley, UK) including treatment with RNase-free DNase set (Qiagen), according to manufacturer’s instructions. For matched miRNA and mRNA expression analysis, expression profiles were generated by Applied Biosystems Clariom D Array, and GeneChip miRNA 4.0, from in total 12 BAV-ND and 12 BAV-D patients. The quality of RNA was analyzed with an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA) and quantity was measured by a NanoDrop (Thermo Scientific, Waltham, MA). The mean RNA integrity number (RIN) was 7.1 ± 0.6. RNA samples with a RIN below 6 were excluded. The Affymetrix GeneChip® Human Exon 1.0 ST array and protocols were used for expression profiling, as previously described [18 (link)]. In total, gene expression was measured in 119 patients (31 BAV-ND, 44 BAV-D, 23 TAV-ND, and 21 TAV-D).

A673/TR/shEF1 cells (Carrillo et al., 2007), which contain a doxycycline (DOX)‐inducible shRNA against EWSR1‐FLI1, were injected subcutaneously in the flanks of immunocompromised NSG (NOD/scid/gamma) mice. When tumors reached an average volume of 180 mm³, mice were randomized and either received 2 mg·mL⁻¹ DOX (Sigma, Darmstadt, Germany) and 5% sucrose in the drinking water (DOX +) or only 5% sucrose (DOX −). Mice were sacrificed after 96 h, and tumors were isolated for RNA and histological analysis. RNA was extracted using the ReliaPrep miRNA Cell and Tissue Miniprep System (Promega, Mannheim, Germany). Knockdown of EWSR1‐FLI1 was confirmed by qRT–PCR (Grünewald et al., 2015). The transcriptome of each tumor (n = 3 for DOX+ and DOX–) was profiled on Affymetrix Clariom D arrays (RIN > 9). Microarray data were normalized on gene level using Signal Space Transformation Robust Multi‐Chip Average and Affymetrix CDF. Three FFPE cores (1 mm) were taken from each xenograft tumor to create a tissue microarray (TMA). Animal experiments were conducted in accordance with the recommendations of the European Community (86/609/EEC), the Government of Upper Bavaria (Germany), and UKCCCR (guidelines for the welfare and use of animals in cancer research).

Samples (derived from SOX10-GFP hES cells) were prepared according to the Affymetrix WT Plus protocol for Gene Chip Whole Transcript Expression Arrays. Briefly 200 ng of high quality total RNA, (RNA integrity number (RIN) greater than 9), was converted to double stranded cDNA with the introduction of a T7 polymerase binding site. This allowed the synthesis of an antisense RNA molecule against which a sense DNA strand was prepared. The RNA strand was digested and the resulting single stranded DNA fragmented and biotin labelled. Along with appropriate controls the labelled fragmented DNA was hybridised to Affymetrix Clariom D arrays overnight using the Affymetrix 640 hybridisation oven; 16 hr with rotation at 60 rpm at 45°C. The arrays were washed and stained according to standard protocols which allowed the introduction of streptavidin-phycoerythrin in order to generate a fluorescent signal from the hybridised biotinylated fragments. The washed and stained arrays were scanned using the Affymetrix 3000 7G scanner with autoloader. The generated CEL files were taken forward for analysis.