Fig. 1. Experimental design.

a Computational framework to select regions and perturbation sites. b Heatmap of motif instances in the assayed regions (left); distribution of the number of motifs perturbed in each region (top right); distribution of the number of regions harboring each motif (bottom right). c Library design; selected regions were included in their wild-type (WT) form, selected motifs were perturbed (by altering the sequence in the predicted motif site) using three perturbation methods individually (PERT single) as well as in combination with other perturbations in selected cases (PERT double). Random sites were perturbed (RAND) and the entire WT sequence was scrambled as negative controls (SCRAM) for each WT sequence. d The designed sequences were synthesized and cloned into the lentiMPRA vector and associated with 15-bp barcodes. ARE antirepressor element, BC barcode. Reporter, EGFP enhanced green fluorescent protein, LTR long terminal repeat, mP minimal promoter, WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. e lentiMPRA libraries were infected into hESCs and following 3 days, we induced neural differentiation via dual-SMAD inhibition and obtained DNA and RNA at seven time points (0, 3, 6, 12, 24, 48, and 72 h). f Association between barcodes and designed sequences, and the number of barcodes observed in DNA and RNA sequencing was determined using MPRAflow²⁸. Differential analysis between WT and PERT activity to determine motif regulatory effect over time was assessed using MPRAnalyze²². Source data are provided as a Source Data file.