Figure 5—figure supplement 2.

(A) Schematic illustrating CRISPR-Cas9-mediated deletion strategy of CRB2-associated PSSE to generate independent ∆CRB2Enh hESC clones with different DNA cleavage products. (B) Flow cytometry analysis for NKX6.1 and PDX1 comparing control and ∆CRB2Enh PP2 cells. Isotype control (ISO) for each antibody is shown in red and target protein staining in green. Percentage of cells expressing each protein is indicated (representative experiment, n = 3 independent differentiations). (C) Immunofluorescent staining for NKX6.1 and PDX1 in control and ∆CRB2Enh PP2 cells (representative images, n = 2 independent slides). Scale bar, 50 μm. (D) mRNA expression of pancreatic transcription factors determined by RNA-seq in control and ∆CRB2Enh PP2 cells. Data are shown as mean of fragments per kilobase per million fragments mapped (FPKM) ± S.E.M. (n = 2 replicates from independent differentiations for control cells ∆CRB2Enh cells represent combined data from two clonal lines with two replicates for each line from independent differentiations. p adj. = 1.00, 1.00, 1.00, 1.00, and 1.00, for comparisons of PDX1, NKX6.1, PROX1, PTF1A, and SOX9, respectively; DESeq2; n.s., not significant). (E) Similarity matrix showing Pearson correlations for normalized transcriptomes (log transformed expression for genes with FPKM ≥1 in ≥1 replicates) in control and ∆CRB2Enh PP2 cells (n = 2 independent differentiations for control cells and for each ∆CRB2Enh clone). See also Figure 5—source data 1.