Genechip scanner 3000

HT29-MTX cells were cultured in 24-well Transwell® (Corning, Corning, NY, USA), and incubated with treatments of MRx0518_LV, MRx0518_HK and MRx0518_SN at a MOI of 100:1 (or equivalent) for 3 h at 37 °C under anaerobic conditions. Cells were washed and lysed, and RNA was isolated from lysate using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was converted to cDNA using a GeneChip™ High Throughput WT PLUS Kit, which was then hybridized to a GeneChip™ Human Transcriptome Array 2.0. Microarray chips were washed and stained using a GeneChip™ Fluidics Station 450 instrument and the GeneChip™ Expression Wash, Stain and Scan kit, and then scanned using a GeneChip™ Scanner 3000 instrument (Thermo Fischer Scientific). Data analysis was carried out using Transcriptome Analysis Console 4.0 software (Thermo Fischer Scientific). Data were normalized using the Robust Multiarray Average algorithm, and fold changes were calculated using the normalized log₂-transformed values of treated cells relative to respective controls. Data were filtered using cut-offs of p < 0.05, fold change of <−1.5 and ≥1.5, and the presence of a gene symbol and coding variants. Pathway analysis was carried out using MetaCore™ (Clarivate Analytics, Philadelphia, PA, USA).

The gene chip was constructed according to the manufacturer’s instructions and previous studies [50 (link),51 (link)]. The gene expression was detected by the Affymetrix scanner (GeneChip^® Scanner 3000, Thermo Fisher Scientific). Based on the RMA algorithm, the data could be analyzed by differential expression gene analysis and another series of follow-up analyses.

To isolate total RNA, sorted cells were flash frozen in PBS immediately after sorting and stored at −80 °C prior to RNA extraction. QIAzol Lysis Reagent (Qiagen) was added to the cells, and RNA was isolated and purified using the RNeasy kit (Qiagen). The concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies). Total RNA was amplified using the GeneChip WT Pico Kit (Thermo Fisher Scientific) generating biotinylated sense-strand DNA targets. The labeled samples were hybridized to human Clariom D arrays (Thermo Fisher Scientific). Washing and staining was performed by the GeneChip Fluidics Station 450 and the scanning was performed using the GeneChip Scanner 3000 (both Thermo Fisher Scientific). All cell populations were generated in triplicate. All data analysis was performed in RStudio. Raw data were normalized using the RMA algorithm implemented in the limma R-package. Adjusted p-values were calculated using the Benjamini-Hochberg method. Data were visualized using ggplot2 and pheatmap R-packages.

RNA was isolated by Rneasy kit (Qiagen, 74106, Hilden, Germany). To purify RNA, Rnase-Free Dnase Set was used (Qiagen, 79254). RNA integrity was measured using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). A 100 ng aliquot of total RNA was linearly amplified. Then, 5.5 µg of cDNA was labeled and fragmented using the GeneChip WT PLUS reagent kit (Affymetrix, 902280, Santa Clara, CA, USA) following the manufacturer’s instructions. Labeled cDNA targets were hybridized to Affymetrix GeneChip Mouse Gene ST 2.0 arrays for 16 h at 45 °C rotating at 60 rpm. The arrays were washed and stained using the Fluidics Station 450 (Thermo Fisher, 00-0079, Waltham, MA) and scanned using a GeneChip Scanner 3000 (Thermo Fisher, 00-0210). Signal intensities were quantified by Affymetrix Expression Console version 1.3.1 (Thermo Fisher). Background correction and quantile normalization were performed to adjust for technical bias, and probe-set expression levels were calculated by the RMA method. After filtering above noise cutoff (p < 0.01), there were 9528 probe-sets tested by linear model. A variance smoothing method with fully moderated t-statistic was employed for this study and was adjusted by controlling the mean number of false positives. QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) was used to identify pathways that were significantly affected by treatments.

RNA synthesis and whole-genome microarray analysis were outsourced to Filgen (Nagoya, Japan). Microarray analysis of miRNAs was performed using the Affymetrix GeneChip miRNA 4.0 Array (Thermo Fisher Scientific) in conjugation with the FlashTag Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific). All reactions and hybridizations were carried out according to the manufacturer’s protocol. These arrays were washed with the GeneChip Fluidics Station 450 (Thermo Fisher Scientific) and scanned with the GeneChip Scanner 3000 (Thermo Fisher Scientific). Differentially expressed miRNAs were analyzed using the Transcriptome Analysis Console 4.0.2 (Thermo Fisher Scientific). To identify differentially expressed genes (DEGs), one-way analysis of variance was conducted, and gene lists were generated by applying a threshold fold change cutoff value of >|2| with a Benjamini–Hochberg false discovery rate (FDR) of < 0.05. We present the differentially expressed transcripts (DETs) in a heatmap with a dendrogram, where the distance metric is Euclidean; the distances between clusters were computed using the complete linkage method to profile gene expression patterns between the tested groups.

Total RNA was extracted from frozen specimens using TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was purified using RNeasy mini kits (Qiagen, Valencia, CA) and RNA integration was checked by gel electrophoresis. The Affymetrix GeneChip Human Genome U133 plus 2.0 (Thermo Fisher Scientific, Santa Clara, CA) was used for microarray experiments. Hybridization and scanning were performed according to the standard Affymetrix protocol. Image scanning was performed using a GeneChip Scanner 3000 (Thermo Fisher Scientific, Santa Clara, CA) with scanned images processed by GeneChip Operating Software and Affymetrix’s Microarray Suite software to generate detection p values. The Robust Multichip Average (RMA) algorithm, which consisted three steps of background adjustment, quantile normalization, and final summarization, was applied for perfect match probe signals within the study^{13 (link)}. For multiple probe sets corresponding to single gene, probe sets were reduced to one per HUGO (Human Genome Organisation) gene symbol by using the most variable probe set measured by inter-quadrant range across all arrays^{14 (link)}.