Fig. 1

MPRA in C2C12 cells identifies sequences with differential enhancer activity. a Summary of MPRA pool design. Cis-acting lincRNAs (right) and their closest protein-coding neighbor (left) are redundantly tiled by 90-bp windows starting every 50 bp. b Core oligo design and experimental overview. Actual oligos flanked by universal primer sites for amplification. Element corresponds to 90-bp genomic sequence, barcode is a 10nt unique identifying tag, and GFP with minimal promoter is inserted after restriction enzyme digestion. c C2C12 expression (TPM) for each gene in the MPRA pool. Red indicates lincRNA, and gray indicates mRNA. d Median sample scatter plot of C2C12 RNA to DNA input control barcode counts (median across replicates used for each barcode, normalized for sequencing depth). e Boxplots of relative signal originating from CMV-promoter-tiling barcodes or oligos tiling five different scrambled versions of the same sequence. Y-axis shows log₂(activity), i.e., log₂(normed RNA counts/normed DNA counts). f Total number of significant regions identified for lincRNAs (red) or mRNAs (gray). g Size distribution of significant regions identified for lincRNAs (red) or mRNAs (gray). h Boxplot of gene body- or promoter-originating oligos from lincRNA loci. The gene body is represented in gray, and the promoter is represented in black. Right, same for mRNA loci. Top illustration: Scheme of TSS-based oligo partitioning. All oligos 1000 bp upstream of a TSS are labeled “promoter” (in black), and the remaining oligos are considered “gene body” (in gray)