Genespring gx 13

The synthesis, labeling, and hybridization of cDNA and cRNA were performed by the Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. The microarray data were analyzed using GeneSpring GX 13.0 software (Agilent Technologies) as described earlier (Daszkowska-Golec et al., 2017 (link)). A gene was considered to be differentially expressed when the level of its expression differed between the analyzed conditions by at least two times (fold change (FC) ≥ 2; P ≤ 0.05 after FDR correction). The annotation of the Agilent Barley Gene Expression Microarray (Agilent Technologies) was performed against IBSC_v2 of barley genome deposited in Ensembl Plants (v. 45). Functional annotation of differentially regulated genes was carried out using Ensembl Plants tools and the IPK Barley BLAST Server as references (https://plants.ensembl.org/index.html, https://webblast.ipk-gatersleben.de/barley_ibsc/). Three biological replications were used for microarray expression analysis (each biological replicate represented leaf of one seedling).

Total RNA was isolated from feeder cells or 3D spheroids on culture day 7 using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Total RNA was amplified and labeled with cyanine-3 (Cy-3) using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s protocol. Cy3-labeled cRNA was hybridized to Mouse Whole Genome Oligo DNA Microarray ver. 2.0 (Agilent Technologies) containing 44,397 features representing 39,429 biological probes using a Gene Expression Hybridization Kit (Agilent Technologies) at 65 °C for 17 h, and scanned with a SureScan Microarray Scanner (Agilent Technologies). Signals in the scanning image were converted into intensity values using Feature Extraction version 11.5.1.1 (Agilent Technologies). Expression raw data from all arrays were analyzed using GeneSpring GX 13.0 software (Agilent Technologies), and global normalization was performed. After the normalization, extremely low intensity probes were excluded, leaving 39,365 probes for analysis. Genes specifically expressed in the 3D spheroids were identified by applying a cutoff of ±1.5-fold and a paired t-test with p < 0.05.

A custom high-density oligo-microarray (8 × 15 K) from the assembled nucleotide European sea bass sequences was designed and printed using the eArray web tool (Agilent). The array comprised 60-oligomer probes for 14,147 different European sea bass annotated sequences. Total RNA (150 ng) from individuals (n = 8 for each intestine segment: AI, MI, and PI) were labeled with cyanine 3-CTP (Low Input Quick Amp Labeling Kit, Agilent), and 600 ng of each labeled cRNA were hybridized to microarray slides that were analyzed with an Agilent G2565C Microarray Scanner according to the manufacturer's protocol. Data were extracted using the Agilent Feature Extraction Software 11.5.1.1. Data analysis of differentially expressed (DE) genes was carried out with the Genespring GX 13.0 software (Agilent). Functional pathway analysis of DE genes was performed with the Ingenuity Pathway Analysis (IPA) software (http://www.ingenuity.com). For each gene, the Uniprot accession of the annotation equivalent for one of the three higher vertebrates model species in IPA (human, rat, or mouse) was assigned.

RNA was extracted from hiPSCs (409B2) and the original HFs (1388) with TRIzol and QIAGEN RNeasy Kit. The total RNA was subjected to cRNA synthesis with 3′ IVT Express Kit (Affymetrix), and the resultant cRNA was fragmented and hybridized to the HG-U133 Plus 2 platform (Affymetrix). After hybridization, GeneChip arrays were washed, stained with GeneChip Fluidics Station 450 (Affymetrix), and detected with Scanner 3000 TG System (Affymetrix) following the manufacturer’s standard protocols. Data analyses were performed with GeneSpringGX 13.0 software (Agilent Technologies).

Microarray results from the nine experimental groups were analyzed by one-way ANOVA (corrected P-value < 0.05, Benjamini-Hochberg), PCA, k-means clustering and correlation analysis with similar entities by means of the Genespring GX 13.0 software (Agilent). PCA eigenvalues were determined by means of Genesis software (release 1.7.7). Optimal number of clusters was determined on the within-group sum of squares calculated by means of the k-means script in R. Ingenuity Pathway analysis used Fisher’s exact test was used to calculate a P-value reflecting the probability that the association between the set of molecules and a given pathway was due only to chance. Threshold of the P-value for association was set to 0.01. In overlapping pathway analysis, settings were selected to guarantee a minimum of 4 common genes between different canonical pathways.