Whole genome expression direct hybridization kit

To analyze global gene expression of SC-β cells, we used a recently described fixation and sorting strategy to isolate NKX6-1+/INS+ SC-β cells from the heterogenous cell clusters (Hrvatin et al., 2014 ). In brief, dispersed and fixed cells were incubated with primary antibodies for 30 min in buffer containing RNasin, washed twice, and then incubated with secondary antibodies in buffer containing RNasin for 30 min each. After antibody staining, cells were sorted by fluorescence activated cell sorting. RNA was extracted with the RecoverAll Total Nucleic Acid Isolation Kit (Ambion). The Illumina TotalPrep RNA Amplification Kit (Life Technologies) was used to make cRNA, which was run on Human HT-12 Expression BeadChips (Illumina) using the Whole-Genome Expression Direct Hybridization kit (Illumina). Chips were scanned on the Illumina Beadstation 500. These SC-β cell microarray data and the previously published hPSC, PH, fetal β and adult β cell data (Hrvatin et al., 2014 ) were imported into the R statistical computing platform using the programming packages lumi and EMA. Samples were analyzed by hierarchial clustering using Pearson’s correlation and Ward linkage. The pattern of clustering was robust to other distance and linkage metrics. Microarray data will be uploaded to publicly available databases.