Prism 6.0h

Statistical significance analysis (GraphPad Prism 6.0h) was performed using one-way or two-way analysis of variance (ANOVA) with the Bonferroni posttest unless otherwise indicated in the figure legends. Bacteriolysis assay data were log transformed prior to statistical analysis (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Statistical analyses were performed using GraphPad Prism 6.0h software (La Jolla, CA, USA) for Mac. Each specific test is indicated in the figure legends. P values of ≤0.05 were considered significant. All data are presented as means ± standard deviations (SDs) from at least three independent experiments.

Off-target site prediction was performed using the CHOP-CHOP tool^{33 (link)}. The potential off-target sites found had at least 2 mismatches with respect to the sgRNA sequence. PCR primers encompassing 4 of these potential off-target sites were designed and the PCR amplicons were purified and sequenced. PCR was performed with Accuprime Pfx (Thermofisher) in the presence of 4 M betaine (Sigma-Aldrich) in 35 cycles, each cycle composed of a denaturing step at 95 °C for 1 min, an annealing step at 60 °C for 1 min and an extension step at 68 °C for 1 min, followed by a final extension at 68 °C for 5 min. Primer sequences are summarized in Supplementary Table 3.
RNA was isolated from GFP⁺ pseudoislet cells using the PicoPure RNA Isolation Kit (Life Technologies). cDNA was synthesized using the Maxima First Strand cDNA synthesis kit (Thermo Scientific) and gene expression was assessed by PCR using the Taqman Gene Expression Mix (Thermo Scientific). Low recovery of mRNA from some pseudoislet preparations after PDX1-CRISPR precluded assessment of some gene expression. Data were analyzed using Prism 6.0 h (GraphPad Software Inc., San Diego, CA). Paired two-tailed t tests were used to indicate statistical significance, and data are presented as mean and standard deviation.

Cells were plated in 384-well plates (1000 cells/well for suspension cell lines and 500 cells/well for adherent cell lines in 30 µl volume). Drugs were serially diluted to the desired concentrations and an equivalent volume of DMSO was added to vehicle control. Ten microliter of the 4× diluted drugs were added to each well. Following 72 h incubation, ATP content was measured using CellTiter-Glo reagent according to manufacturer’s instructions (Promega, CellTiter-Glo Luminescent Cell Viability Assay), and analyzed by SpectraMax luminometer (Molecular Devices). IC₅₀ values, concentrations required to inhibit proliferation by 50% compared to DMSO treated cells, were calculated using Prism 6.0 h (Graphpad Software).