Fluorolink streptavidin cy3

Total RNA from Shp2^hep−/− and WT mice liver was prepared with RNeasy mini kit (Qiagen Cat # 74104). Labeled cRNA was prepared from 500 ng RNA using the Illumina® RNA Amplification Kit from Ambion (Austin, TX, USA). The labeled cRNA (750 ng) was hybridized overnight at 58°C to the Sentrix Mouse -8 Expression BeadChip (>23,000 gene transcripts; Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. BeadChips were subsequently washed and developed with fluorolink streptavidin-Cy3 (GE Healthcare). BeadChips were scanned with an Illumina BeadArray Reader. The microarray data have been deposited in the Gene Expression Omnibus (GEO) under the accession number of GSE51860. The gene expression data (GSE20599) for FXR^−/−/SHP^−/− DKO mice at 5 weeks of age were downloaded from the GEO (Anakk et al., 2011 (link)), processed with BeadStudio software and quantile normalized. The data (GSE29426) for FGF15/19-treated mice were downloaded from GEO (Potthoff et al., 2011 (link)) and processed with MAS5 algorithm (Affymetrix). Probes were filtered with detection p-value > 0.01 (for Shp2^hep−/− and FXR^−/−/SHP^−/− DKO data) or with ABS call (for FGF15/19 data) before further analysis. Transcripts shared between datasets were used for K-means clustering with Cluster 3.0 software. Heat maps were generated with Java TreeView. GO analysis was performed with DAVID v.6.7 program.