RNA-Seq Analysis of Human Islets and Pseudoislets

Aliquots of cultured human islets and their respective pseudoislets were used for RNA extraction using NucleoSpin® RNA XS (Macherey-Nagel, Düren, Germany). RNA integrity (RIN ≥ 9) was measured with Bioanalyzer 2100 (Agilent Technologies). Sequencing was performed as described previously at the Center for Molecular and Cellular Bioengineering (Andreas Dahl, CMCB, Technical University Dresden, Germany)^{45 (link)}. In short, mRNA was isolated from 120 ng RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module. After fragmentation, the samples were subjected to the workflow for strand-specific RNASeq library preparation (Ultra Directional RNA Library Prep II, NEB) and 75 bp single read sequencing was performed on Illumina NextSeq500. After sequencing, FastQC was used to perform quality control. Differential expression between islets and pseudoislets was tested with DESeq R package (v.1.10.1). The complete analysis is available in Supplementary File 2 and under GEO code GSE133903.