Fig. 2

QeFS provides quantitative enhancer activity data consistent with RT-qPCR data. A, B QeFS measurements recapitulate the quantitative effects of previously published binding site knockouts in two different mesodermal enhancers. Asterisks within bars represent statistically significant differences (adjusted P-value < 0.05, Conover-Iman post-hoc test with Benjamini-Hochberg correction) from wildtype enhancer activity; asterisks with horizontal lines indicate significant pairwise differences between mutant constructs. C, D Reporter gene expression measured by RT-qPCR from whole embryo RNA in biological triplicates is generally concordant with QeFS measurements. Significance assessed by paired-sample t-test of Cq values. E Reproducibility of QeFS data. QeFS expression levels from week 1 were highly correlated with those from week 2 for enhancers in the mef2:CD2- context (Spearman’s ρ = 0.95). F Validation of QeFS data. Effects of mutations on QeFS-measured expression levels of enhancers in mef2:CD2- cells were correlated with effects of the same mutations measured by whole embryo qRT-PCR in biological triplicates (Pearson’s R = 0.525). Green quadrant represents mutants for which both methods found increased expression; red quadrant highlights mutants for which both methods found decreased expression relative to wild type. Error bars in all panels show s.e.m. of measured fold change