Feature extraction 9

Copy number analyses were performed by CMA using an Agilent customer design oligonucleotide 8 × 60 K CytoScan^® gene chip (ID 040427). DNA labeling and hybridization were carried out according to manufacturer’s recommendation. Scanned images were analyzed by Feature Extraction 9.5.3 software (Agilent Technologies, Santa Clara, CA, USA), and the extracted data were processed using the Agilent Genomic Workbench 7.0 program (Agilent Technologies, CA, USA). The CMA findings were described based on the reference genome version of GRCh37, following the latest guideline of An International System for Human Cytogenomic Nomenclature (ISCN2020).

Labeling was performed using the Agilent LowInput QuickAmp Labeling Kit One-Color (5190 − 2305; Agilent Technologies).The SurePrint G3 Rat GE 8 × 60K Kit were used (G4853A, Agilent Technologies). Briefly, first-strand cDNA synthesis was performed using an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site. The cDNA was used as a template to generate Cy3-labeled cRNA that was used for hybridization. After purification and fragmentation, aliquots of each sample were hybridized to Agilent Oligo Microarrays (G2534-60014, Agilent Technologies). After hybridization, each array was sequentially washed and scanned by Agilent microarray scanner. Arrays were individually visually inspected for hybridization defects, and quality control procedures were applied as recommended by the manufacturer of the arrays. For array readout, Agilent Feature Extraction 9.5.3 Software was used, and microarray data were imported, log2-transformed and quantile normalized using robust multi-array average (RMA), and expression levels were summarized on a transcript level using average gene expression values of the replicated probes, as recently described [19 (link)]. Differential gene expression was calculated using the moderated t-test as described by Ritchie et al. [20 (link)] and implemented in the R/Bioconductor package limma.

All microarray hybridizations were performed according to the manufacturer’s instructions in the One-Color Microarray-Based Gene Expression Analysis manuals with only one modification. Instead of hybridizing the recommended amount of 1.6 μg Cy-3 labelled RNA to the array, we used 2.4 μg to boost the signal because the hemoglobin RNA removal step was not employed (this increase was used only after careful analysis of its reproducibility and results as compared with the standard protocol). The fluorescently labelled RNA was hybridized to the microarray at 65 °C in a rotating oven. After 17 hours, the arrays were washed consecutively in solutions of 6X SSPE with 0.005% N-lauroylsarcosine and 0.06X SSPE with 0.005% N-lauroylsarcosine for 1 min each at room temperature. This wash was followed by a final 15 s wash in acetonitrile. Microarrays were then imaged on an Agilent microarray scanner and extracted using Agilent Feature Extraction 9.5.

Array CGH was performed on Agilent’s SurePrint G3 Human CGH Microarray Kit 2x400K (Design ID021850, Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Scanning of the hybridized arrays was carried out using the Agilent High-Resolution Microarray scanner. Raw data were processed by the Feature Extraction 9.5 (Agilent) software and normalization was performed using the default settings. Feature Extraction files were imported into Genomic Workbench 7.0 (Agilent Technologies) for visualization and analysis. After diploid centralization and GC correction, aberrations were called using the ADM2 algorithm with a threshold setting of 20, centralization on with threshold of 25 and an aberration filter min Probes = 5 and minAvgAbsLogRatio = 0.35 for amplifications and deletions [13 (link)].

Oligonucleotide array CGH was performed using the Agilent Human Genome CGH Microarray Kit 44 K®. This microarray consisted of more than 44,000 oligonucleotide probes that spanned both coding and non-coding regions. The coverage of the human genome was made with an average spatial resolution of 75,000 pair bases.
The patient’s DNA as well as a reference DNA was fragmented by heat at 95 °C for 20 min. Each fragmented DNA product was labeled by random priming using either ULS5 or ULS3. After column-purification, probes were denatured and pre-annealed with 5 μg of human Cot-1 DNA, 10 μl of CGH Blocking agent and 55 μl of hybridization buffer. Hybridization was performed at 65 °C during 24 h. The microarray was washed, scanned and analyzed with Agilent Feature Extraction® 9.1 software. Results were interpreted using DNA analytics® 4.5 software. Only imbalances involving three or more adjacent probes were held. The identification of probes with a significant gain or loss was based on the log² ratio plot deviation from 0 with cutoff values of 0.5 to 1, and − 0.5 to − 1, respectively.

This was performed following protocols from Agilent. Briefly, 600 ng of each labeled cRNA was fragmented and then mixed with hybridization buffer using the Agilent gene expression hybridization kit. These were applied to an ovine 8 X 15 K array slide (Agilent 019921), containing 8 arrays with 15,208 oligomers with a length of 60 bases and hybridized at 65°C for 17 h at 10 rpm. The arrays were washed, dried, stabilized, and scanned with an Agilent G2505B 2 dye scanner at the Interdisciplinary Center for Biotechnology Research at the University of Florida. Features were extracted with Agilent Feature extraction 9.1 software. Microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE99497.

This was performed following protocols from Agilent. Briefly, 600 ng of each labeled cRNA was fragmented and then mixed with hybridization buffer using the Agilent gene expression hybridization kit. These were applied to sheep 8 × 15 K array slides (Agilent 019921), containing 8 arrays with 15,208 oligomers with a length of 60 bases and hybridized at 65°C for 17 h at 10 rpm. A total of 3 slides (24 arrays) were employed per each brain region (six gestational ages times four replicates per gestational age). The arrays were washed, dried, stabilized, and scanned with an Agilent G2505B 2 dye scanner at the Interdisciplinary Center for Biotechnology Research at the University of Florida. Features were extracted with Agilent Feature extraction 9.1 software.