Extended Data Fig. 1: Design and reproducibility of ExP STARR-seq.
From: Compatibility rules of human enhancer and promoter sequences
a. ExP STARR-seq reporter construct (pA = polyadenylation signal; purple = promoter sequencing adaptors; angled = spliced sequence; trGFP = truncated GFP open reading frame with start and stop codon; BC = 16 bp N-mer plasmid barcode; red = enhancer sequencing adaptors) and 1000x1000 K562 library contents. b. Correlation of ExP STARR-seq expression between biological replicate experiments, calculated for individual enhancer-promoter pairs with unique plasmid barcodes. Axes represent the average STARR-seq expression (RNA/DNA) of individual biological replicates. Density: number of enhancer-promoter plasmids. c. Fraction of remaining enhancer-promoter plasmids passing DNA (>25) and RNA (>1) threshold (y-axis) with downsampling of sequencing reads (x-axis). d. Distribution of plasmid barcodes per enhancer-promoter pair, red dotted-line is threshold of two plasmid barcodes. e. Correlation between virtual replicates, formed by sampling two nonoverlapping groups of three plasmid barcodes from pairs with at least 6 barcodes, and averaging log2(RNA/DNA) within groups. f. Correlation between virtual replicates as in (c) for increasing numbers of plasmid barcodes per pair in virtual replicates. g. DNase-seq, H3K27ac ChIP-seq, and PRO-seq (RPM) by increasing quartile of autonomous promoter activity and average enhancer activity in ExP STARR-seq (n = 800). Box: median and interquartile range (IQR). Whiskers: +/− 1.5 x IQR. h. Activation in ExP STARR-seq (expression versus genomic controls in distal position) of GATA1 and HDAC6 promoters by eHDAC6 (chrX:48641342-48641606). Ctrl = activity of promoters with random genomic controls in enhancer position. Error bars: 95% CI across plasmid barcodes. n = 7 (GATA1-ctrl), 381 (HDAC6-ctrl), 4 (eHDAC6-GATA1), 37 (eHDAC6-HDAC6). i. Average enhancer activity (STARR-seq expression of plasmids containing a given enhancer averaged across all promoters) of enhancer sequences derived from random genomic controls (n = 87), accessible elements (n = 725), and genomic enhancers validated in CRISPR experiments (n = 89).
