RNA-seq profiling of sorted pancreatic beta cells

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.