Fig. 5

RNA-seq gene expression analysis of dCas9 3A3L treated and control myoepithelial cells. a PCA analysis of RNA-seq data using all transcripts from early passage (blue), dCas9 3A3LΔ targeted (purple) and dCas9 3A3L targeted (orange) primary myoepithelial cells from donor 1, 10 days post-transfection targeting HIC1, RASSF1, PTEN and CDKN2A (including magnetic sorting at day 2). Each circle represents an individual biological replicate (n = 3). b PCA analysis using only data from transcripts which are differentially expressed between dCas9 3A3L and 3A3LΔ targeted cells as identified by DESeq2 (p < 0.001; with BH correction); data are shown as early passage (blue), dCas9 3A3LΔ targeted (purple) and dCas9 3A3L targeted (orange) cells. Each circle represents an individual biological replicate (n = 3). c GO analysis of transcripts which are differentially expressed between dCas9 3A3L and 3A3LΔ targeted cells. GO terms associated with transcripts highly expressed in dCas9 3A3L targeted cells (left panel) and 3A3L∆ targeted cells (right panel) are shown. P value was calculated using Cytoscape (v. 3.4.0) and the add-in package Cluego (v. 2.2.5), which utilises the KEGG pathway database. d, e Gene expression of transcripts associated with d p16 signalling and e p14 signalling from the RNA-seq data 10 days post-transfection in primary myoepithelial cells from donor 1. Data is shown as fold change of gene expression from 3A3L∆ (purple) and 3A3L (orange) compared to average RPKM data from early passage cells (dotted line; mean ± SEM, n = 3). Significance is from DESeq2 analysis: *p < 0.001 3A3L compared to 3A3LΔ targeted cells; ^$ p < 0.001 3A3L or 3A3L∆ compared to early passage