Genechiptm wt plus reagent kit

The total RNA from cells was isolated using peqGOLD TriFast™ according to the manufacturer’s protocol. RNA samples were purified using the RNA Clean-Up and Concentration Micro Kit, RNA concentration was determined using spectrophotometry (NanoDrop 8000), and quality control was performed using an Agilent 2100 Bioanalyzer. Microarray analysis was carried out using individual RNA samples (n = 4), which were processed following the manufacturer’s instructions of the GeneChip^TM WT PLUS Reagent Kit (Thermo Fisher Scientific) and hybridized with GeneChip^TM Clariom D Rat Arrays (Thermo Fisher Scientific). Quality control of the hybridizations and data analysis were performed using Transcriptome Analysis Console (Thermo Fisher Scientific). The data were normalized using the Robust Multi-chip Analysis (RMA) algorithm.
To identify significantly differentially expressed genes (q–value < 0.05, fold change ≥ 1.3-fold), one-way ANOVA (with Bayes estimation) was performed, where p-values were corrected for multiple testing using Benjamini Hochberg adjustment. All data from transcriptome analyses are provided in Table S1. Significantly differentially expressed genes were further subjected to in silico pathway analysis using the Ingenuity Pathway Analysis software (Ingenuity Systems, Inc. Redwood City, CA, USA). This enrichment analysis was based on all annotated rat genes in the database.

Colon tissues were disrupted using a Precellys 24 (Bertin technologies) and total RNA from colon tissue was extracted using a Nucleospin® RNA kit (Macherey-Nagel) according to manufacturer’s instruction. 100 ng of total RNA per sample were used as input for the Clariom-S-mouse microarray (Thermo Fisher Scientific) analysis. Target preparation was performed using the Gene Chip^TM WT PLUS Reagent Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Hybridization was performed in a Gene Chip Hybridization Oven 645 for 16 h at 45 °C. Gene chips were scanned using the Gene Chip Scanner 7 G. The Clariom-S-mouse array was in a 169 format, for which the Fluidics Protocol FS450-0007 was used. Array-Quality Control was done using the Affymetrix Expression Console or Transcriptome Analysis Console (TAC) Software. Z score transformation and heat cluster data analysis was performed using InstantClue^{59 (link)}.

An additional RNA purification step was done by using RNeasy MinElute Cleanup Kit (Quiagen, Netherlands; 74204), according to the manufacturer instructions. RNA quality was dissected via use of a Bioanalyzer (2100 Bioanalyzer, Agilent, CA, USA; Supplemental Figure S1A–D). Detection of RNA was performed by microarray, according to the manufacturer’s protocol (CLARIOM D ARRAY, MOU, Thermo Fisher Scientific, Waltham, MA, USA). Shortly, biotin-labeled ss-cDNA was synthesized from total RNA with a GeneChipTM WT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA), and subsequently hybridized using Clariom D mouse arrays (Thermo Fisher Scientific, MA, USA) and GeneChip Fluidics station 450 (Thermo Fisher Scientific, Waltham, MA, USA). Hybridized mRNA chips were washed and scanned by the Affymetrix GeneChip Scanner 7G (GeneChip Command Console 3.1 software). Data validation was performed with a Transcriptome Analysis console (TAC 4.0; applied biosystems; Thermo Fisher Scientific, Waltham, MA, USA). Differentially expressed genes were displayed through fold change (up-regulated > 2.0; down-regulated < −2.0), together with a p value < 0.05. Functional annotation of the genes was done with the Consensus Path DB-mouse (cpdb.molgen.mpg.de; MM11; [10 (link)]) to find enriched pathways.