3d gene scanner

Total RNAs from cultured intestinal organoids were extracted using the RNeasy Plus Mini Kit and QIAshredder (QIAGEN). Microarray analysis was conducted by Toray Industries (Tokyo, Japan). In brief, extracted total RNA was checked with a Bioanalyzer (Agilent Technologies) and labeled with Cy5 and Cy3. The labeled RNAs were hybridized onto a Human Oligo chip 25k (Toray Industries). After stringent washing, the fluorescent signals were scanned with a 3D-Gene Scanner (Toray Industries) and analyzed using the 3D-Gene Extraction software (Toray Industries). The raw data for each spot were normalized by subtraction of the mean background signal intensity determined from the signal intensities of all blank spots with 95% confidence intervals. The relative expression level was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. All data were submitted to the GEO database, under the accession number GSE103634.

Total RNA was extracted from the cells with a QIAGEN RNeasy Mini kit (QIAGEN, Hilden, Germany), as described previously^{37 (link)} according to the manufacturer’s protocol. Genome-wide expression profiling was performed using a 3D-Gene Scanner with 3D-Gene Oligo chip 24 k (Toray Industries, Inc., 23,522 distinct genes) and the supplier's protocol. Hybridization signals were scanned 3D-Gene Scanner (Toray Industries) and processed by 3D-Gene Extraction software (Toray Industries). The raw data of each spot was normalized by subtraction with a mean intensity of the background signal determined by all blank spots' signal intensities of 95% confidence intervals. The raw data intensities greater than 2 standard deviations (SD) of the background signal intensity were considered to be valid. cDNA microarray was performed using pooled RNA samples from 2 independent experiments for each condition. Second cDNA microarray was performed using different sets of samples for each condition and major up-regulated genes including IFIT3 were confirmed. Data analysis and functional analysis was performed by Gene Ontologies and KEGG pathway analysis. GSEA was performed according to the instructions.

Microarray of EV miRNAs was conducted in Toray Industries using 3D‐Gene device. Briefly, total RNAs were extracted from 1 × 10¹¹ particles of L‐s and H‐s CTL EVs using TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. The total RNAs were labelled using a 3D‐Gene miRNA labelling kit (Toray Industries). Labelled RNAs were hybridized onto a 3D‐Gene Mouse miRNA Oligo chip (Toray Industries). The annotation and oligonucleotide sequences of the probes conformed to miRBase (http://microrna.sanger.ac.uk/sequences/). After stringent washes, fluorescent signals were scanned using a 3D‐Gene Scanner (Toray Industries) and analyzed using 3D‐Gene Extraction software (Toray Industries). The relative expression level of the given miRNAs was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The data were globally normalized per array, such that the median signal intensity was adjusted to 25.

Transcriptome analysis using the 3D‐Gene Human Oligo Chip 25 k (Toray Industries Inc., Kanagawa, Japan) and subsequent data analysis were performed by Toray Industries Inc. SW480 cells were treated with vehicle control DMSO (n = 3) or 10 μm NC114 (n = 3) for 8 h, and total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The purified RNA was labeled with Cy5 using the Amino Allyl MessageAMP II aRNA Amplification Kit (Thermo Fisher Scientific). The Cy5‐labeled aRNA pools were mixed with hybridization buffer and hybridized for 16 h. The hybridization signals were obtained using a 3D‐Gene Scanner (Toray Industries Inc.) and processed with 3D‐gene extraction software (Toray Industries Inc.). Detected signals for each gene were normalized by a global normalization method. The median of the signal intensity was adjusted to 25. The downregulated and upregulated genes (1.5‐fold or higher changes) were analyzed for enrichment based on Gene Ontology (GO) terms using genespring gx software (Agilent Technologies, Santa Clara, CA, USA).

Total RNA of cell lines and organoids was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, Germany), at day 14 (P2) for Cx-1, d25 (P3) for Cx-2, and d10 (P1) for Cx-3. These RNA samples were subjected to the TORAY 3D-gene analysis service using the 3D-Gene Human Oligo chip 25K (TORAY, Tokyo, Japan). Total RNA was amplified and labeled with Cy5, then hybridized with a 3D-Gene chip. Signals were detected on a 3D-Gene scanner (TORAY) and normalized according to a global normalization method in which the median value of the detected signal intensities was adjusted to 25. With regard to SCJ markers, genes without corresponding probes and with an average signal intensity lower than 20 are listed in Table S2. Differentially expressed genes between three cell lines and organoids were extracted from the microarray data. Genes with normalized signal intensities less than 20 in more than three samples were excluded from the analysis. GeneSpring GX (Ver.14.9.1, Agilent Technologies, Santa Clara, CA, USA) was used to generate a heat map. Metascape [37 (link)] was used to identify enriched KEGG pathways of the focused clusters. A p-value < 0.05 was considered statistically significant. Microarray data were deposited to GEO accession: GSE138554.

Extracted total RNA was labeled with Hy5 using the miRCURY LNA Array miR labeling kit (Exiqon, Vedbaek, Denmark). Labeled RNAs were hybridized onto 3D-Gene Human miRNA Oligo chips (Toray, Kamakura, Japan). Annotation and oligonucleotide sequences of the probes conformed to the miRBase database (http://microrna.sanger.ac.uk/sequences/). After stringent washing, fluorescent signals were scanned with the 3D-Gene Scanner (Toray) and analyzed using 3D-Gene Extraction software (Toray). The microarray data is available on NCBI’s GEO database (Accession number: GSE81828).
The raw data for each spot were normalized to the mean intensity of background signals determined by all blank signal intensities at a 95% confidence interval. Measurements of spots with signal intensities greater than 2 standard deviations (SD) of the background signal intensity were considered to be valid. Relative levels of expression of a given miRNA were calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. Normalized data were globally normalized on each array, such that the median of the signal intensity was adjusted to 25.