Beadstation 500

Total RNA was isolated using Qiagen RNeasy kit (Qiagen), and 500 ng of total RNA was used to synthesize dscDNA. Biotin-labeled RNA was generated using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre), according to the manufacturers’ instructions, and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina), which contains more than 47,000 probes derived from the NCBI RefSeq Release 38 and other sources, at 58°C for 16 h. The chip was washed, stained with streptavadin-Cy3, and scanned with the Illumina BeadStation 500 and BeadScan. Using the Illumina's GenomeStudio software, we normalized the signals using the “cubic spline algorithm” that assumes that the distribution of transcript abundance is similar in all samples, according to the method proposed by Workman et al. [30 ]. The background signal was removed using the “detection p-value algorithm” to remove targets with signal intensities equal or lower than that of irrelevant probes (with no known targets in the human genome but thermodynamically similar to the relevant probes). Common and unique sets of genes and enrichment analysis were performed using the MetaCore Software (Thompson Reuters) as previously reported [14 (link)]. The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE79548).

Clusters from several independent batches made from a subset of cell lines were dispersed with TrypLE, fixed in 4% PFA supplemented with RNasin (VWR; PAN2615) for 30 min on ice, stained with NKX6-1 and INS primary antibody in RNasin-containing buffer for 30 min on ice and stained with appropriate Alexa Fluor-488 and -647 secondary antibodies diluted in RNasin-containing buffer for 30 min on ice. INS+/NKX6-1+ cells were sorted using an FACSAria (BD Biosciences) and RNA extracted by first incubation sorted cells in digestion buffer (RecoverAll Total Nucleic Acid Isolation Kit; Ambion; AM1975) at 50 °C for 3 h then by following the instructions from the manufacturer. cDNA and cRNA was generated with Illumina TotalPrep RNA Amplifcation Kit (Life Technologies; AMIL1791). cRNA was hybridized to Human HT-12 Expression BeadChips (Illumina) with the Whole-Genome Expression Direct Hybridization kit (Illumina) and chips read with Illumina Beadstation 500. Data were analysed in GenomeStudio (Illumina) with background subtraction and rank-invariant normalization. Previous published data for HUES8 SC-β cells, undifferentiated HUES8, fetal β-cells and adult β-cells was also included in data analysis17 (link)22 (link). Hierarchical clustering was performed using Pearson's correlation and Ward linkage.

Microarray analysis was performed and analyzed at the Stanley S. Scott Cancer Center's Translational Genomics Core at LSUHSC. Total RNA was isolated using Qiagen RNeasy kit (Qiagen), and 500 ng of total RNA was used to synthesize dscDNA. Biotin-labeled RNA was generated using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre), and hybridized to the HumanHT-12 v4 Expression BeadChip (Illumina) at 58°C for 16 h. The chip was washed, stained with streptavadin-Cy3, and scanned with the Illumina BeadStation 500 and BeadScan. Using the Illumina's GenomeStudio software, we normalized the signals using the “cubic spline algorithm” that assumes that the distribution of transcript abundance is similar in all samples. The background signal was removed using the “detection p-value algorithm” to remove targets with signal intensities equal or lower than that of irrelevant probes (with no known targets in the human genome but thermodynamically similar to the relevant probes). The microarray experiments were performed twice for each group and the average values were used for analysis. Common and unique sets of genes and enrichment analysis were performed using the MetaCore Software (Thompson Reuters). The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE90038).