Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
This study investigates the roles of Ngn3 and NeuroD1 in pancreatic endocrine differentiation. Using a new mouse model, we mapped NGN3 binding sites by CUT&RUN and demonstrated its pioneering role in making chromatin accessible by scATAC-seq. According to conditional knock-out and over-expression experiments and scRNA-seq, we domonstrated that NGN3's pioneering function was dose tolerance, with low doses sufficing, while NeuroD1, as a conventional TF, has no significant pioneer effects under normal phenomena. But when NGN3 was absent, NeuroD1 can alternatively acted as a pioneer factor, and sequential expression of NeuroD1 ahead of Ngn3 predominantly drives α-cell generation.
Overall design
Six types of datasets were generated in this project. 1. CUT&RUN of Ngn3-flag, NeuroD1 or P300 in Ngn3low or Ngn3high cells isolated from E13.5 or E14.5 fetal pancreas. CUT&RUN of NeuroD1 in GFP+ cells isolated from E15.5 Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice. 2. 10x genomics snATAC-seq of endocrine or pancreatic cells collected from Ngn3CreER;RossaRFP or Pdx1Cre;RossaRFP mice. 10x genomics snATAC-seq of Epcam+ cells collected from Neurod1KO or Ngn3Cre;Ngn3fl mice and their corresponding WT groups (CKO/OE and WT were distinguished by gender). 3. ATAC-seq of Ngn3low or Ngn3high cells in E13.5 fetal pancreas. ATAC-seq of the E13.5 GFP+ cells isolated from Ngn3KI mice, E15.5 or E17.5 Venus+ cells isolated from Neurod1KO mice, E15.5 GFP+ cells isolated from Pdx1CreER;Ngn3fl;Neurod1OE, Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice. 4. 10x genomics scRNA-seq of the Epcam+ cells isolated from E14.5 Ngn3Cre;Ngn3fl, E14.5 or E17.5 Ngn3Cre;Ngn3OE and E15.5 Neurod1KO mice and their corresponding control WT groups (CKO/OE and WT were distinguished by gender). 5. Smartseq3 scRNA-seq of the E12.5 or E15.5 GFP+ cells isolated from Pdx1CreER;Ngn3fl;Neurod1OE, Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice, and Epcam+ cells isolated from WT mice. 6. Smartseq2 scRNAseq of Ngn3flagGFPlow, Ngn3flagGFPhigh and GFP+ cells isolated from Ngn3KI mice cells.
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