Massively parallel reporter assay (MPRA)

Enhancers were synthesized by Agilent as an oligonucleotide pool. Each enhancer consisted of two 230 bp ssDNA oligos with 20 bp 3′ overlap. Each oligonucleotide’s 5′ end had a 20 bp primer-binding site. Oligonucleotides within the pool were annealed and 3′ ends were extended by PCR to create a library of 400 bp enhancers flanked by 20 bp priming sites. NotI/AscI-restriction sites were added to enhancers in a second round of PCR. The enhancer library was then digested, size selected, and ligated into the multiple cloning site of a self-complementary AAV plasmid containing a minimal MLC2v promoter-mCherry-NotI/AscI-polyA sequences. The ligation product was electroporated into Agilent SURE Electrocompetent cells following manufacturer recommendations, spread onto agar plates, and cultured overnight. Approximately 900,000 colonies were harvested and pooled for plasmid maxi-prep. The enhancer library was packaged into AAV as described above. P0 wildtype CFW mouse pups (n = 28) were injected subcutaneously with 50 µl saline containing containing 2E11 vg. Hearts were harvested at P7 and RNA was isolated from homogenized ventricular apexes via TRIzol phase extraction and reverse transcribed using a primer recognizing the start of the polyA sequence. NGS adapters and unique indexes were added to each sample in subsequent rounds of PCR amplification. Untransduced AAV DNA from the library pool was also prepared in the same fashion for sequencing in triplicate. Indexed samples were pooled and paired-end (2 × 150 bp) sequenced on a NextSeq500. After removal of adapters, reads were aligned to the mouse genome, keeping only mates that produced concordant alignments between 395 and 405 bp. On average, each sample contained ~5M alignments passing these criteria. The number of reads for each enhancer in each sample was determined using BedTools⁵⁰. The average number of reads for each enhancer within the untransduced AAV DNA was then calculated, and enhancers present at low frequencies (<5 RPM) were excluded. The majority (>90%) of enhancers were successfully created and were detected above the 5 RPM threshold. Read numbers for RNA samples were acquired using the same method. RNA:DNA ratio for each region was calculated and used as a readout of enhancer activity.