Single-cell RNA sequencing of human embryonic stem cell-derived pancreatic cells enriched for early endocrine progenitors using a novel dual NEUROG3-reporter. Single-cell RNA sequencing of human embryonic stem cell-derived pancreatic cells enriched for early endocrine progenitors using a novel dual NEUROG3-reporter

Human embryonic stem cells with one allele of the NEUROG3 gene genetically modified into a fusion gene NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS were differentiated into the pancreatic lineage until Stage 4 Day 1 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017). The cells were index-sorted according to their GFP expression to enrich for low GFP and high GFP cells, and deep single-cell RNA-seq was performed using the plate-based Smart-seq2 platform (Picelli et al. 2014).

将携带NEUROG3基因一个等位基因且经遗传修饰为融合基因（fusion gene）NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS的人胚胎干细胞（human embryonic stem cells），采用经Petersen等（2017）修改的Rezania等（2014）体外分化方案（in vitro differentiation protocol），分化至胰腺谱系的第4阶段第1天。随后根据细胞的GFP（Green Fluorescent Protein）表达水平进行索引分选（index-sorted），以富集低GFP与高GFP表达细胞，并采用基于平板的Smart-seq2平台（Smart-seq2 platform，Picelli等，2014）完成深度单细胞RNA测序（deep single-cell RNA-seq）。