title: 10x scRNA-Seq of cells in stage 4 of protocol  for differentiating islet-like organoids from human embryonic stem cells  (H9)
design: Cells used for single-cell RNA-sequencing (scRNA-seq) were dissociated at stage 4 from hESC(H9)-derived islet organoids uding our protocols. Single-cell libraries were prepared with the Chromium Single Cell 3 Reagent Kits v2 (10x Genomics) . Briefly, sorted cells in suspension were first prepared as gel beads in emulsion (GEMs) on Single Cell 3 Chips v2 (10x Chromium) using the Chromium Controller (10x Genomics). Barcoded RNA transcripts in each single cell were reverse transcribed within GEM droplets. cDNA was purified with DynaBeads MyOne Silane beads (Invitrogen) and then amplified for subsequent library construction. Sequencing libraries were prepared by fragmentation, end-repair, ligation with indexed adapters, and PCR amplification using the Chromium Single Cell 3 library kit v2 (10x Genomics). Nucleic acid was cleaned up after each step using SPRIselect beads (Beckman Coulter). Libraries were then quantified by Qubit and real-time quantitative PCR on a LightCycler 96 System (Roche).