title: 10x scRNA-Seq of cells in stage 5 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 5 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).