strain: C57Black6 gender: male tissue: intestinal organoids exposed to: butyrate
Extracted molecule
total RNA
Extraction protocol
Cultured organoids were pooled, approximately 300 individual organoids per sample, and RNA was isolated using Trizol (Invitrogen, Breda, the Netherlands), followed by purification using the RNeasy Kit (Qiagen). Samples were assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and NanoDrop Spectrophotometer (NanoDrop, Wilmington, DE).
Label
biotin
Label protocol
Biotinylated cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit (Ambion, Inc., Austin, TX, U.S.A.) according to the manufacturer’s specifications with an input of 200 ng total RNA.
Hybridization protocol
Total mRNA was amplified, biotinylated and randomly hybridized to MouseRef-8 v2 Expression bead arrays (Illumina, Inc., San Diego, CA, U.S.A.) following standard Illumina hybridization protocol.
Scan protocol
Scanning was performed on the Illumina iScan (Illumina, Inc., San Diego, CA, U.S.A.).
Description
9231425044_E
Data processing
Data were extracted using GenomeStudio. Quality control and normalization of microarray data was performed in ArrayAnalyzer (www.arrayanalyzer.org). Non-expressed genes were removed by filtering on detection value (p value ≥0.99 in at least one sample). This resulted in gene expression values for 25,697 probes. For differential expression analysis Limma software was used. Subsequently, expression data of the transcriptomics were log-transformed (base 2). Probes were used for biological interpretation if the p value threshold of <0.01 was passed and if the fold change between two conditions was |1.5|.
