Genomic DNA and RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen, 80204) with vortexing to lyse cells.  Libraries were electroporated in NEB 3020K electro-competent cells. Plasmids from cells were bottlenecked and extracted using the ChargeSwitch MidiPrep kits per manufacture's instructions. MPRA amplicon libraries from genomic DNA were generated in two steps of PCR amplification with OneTaq (NEB, M0482S). Approximately 10 µg of genomic DNA input was used per replicate. For the first low-cycle PCR (PCR1), gDNA was mixed with 200 µl 2× OneTaq master mix, 2 µl 100 µM indexed P5 primer (rand-P5-E##), 2 µl 100 µM P7-pLSmp-assUMI-gfp primer, and water to a final volume of 400 µl. This mix was prepared for each replicate and then split over 16 wells for PCR. Cycling parameters were: 2 min at 95 °C, followed by three cycles of 20 s at 95 °C, 30 s at 55 °C, and 2 min at 72 °C, with a final extension of 5 min at 72 °C, followed by a 4 °C hold. Primer P7-pLSmp-assUMI-gfp contains ten random nucleotides (Ns) to serve as a pseudo-UMI (hereafter referred to as UMIs for brevity) to correct for PCR jackpotting. Reactions were pooled for each replicate, cleaned up with AMPure XP beads (Beckman Coulter, A63881) at 1.8×, and eluted in 97 µl of 10 mM Tris pH 8.0. A second round of PCR (PCR2) was performed for each replicate with primers P5 and P7, which amplified the library for sequencing without adding additional UMIs. The entire 97 µl of eluate from PCR1 was used as input and combined with 100 µl OneTaq master mix, 1 µl 100× SYBR Green (Thermo Fisher, S7563), 1 µl 100 µM P5 primer, and 1 µl 100 µM P7 primer. This mix was split over 8 reactions for each replicate. Libraries were amplified and monitored in real-time by qPCR with 2 min at 95 °C, followed by cycles up to the qPCR inflection point consisting of 15 s at 95 °C, 20 s at 60 °C, and 2 min at 72 °C. Libraries were then pooled by replicate and cleaned up with AMPure XP beads (Beckman Coulter, A63881) at 1.8×.  Amplicon libraries from RNA were obtained by first DNase-treating the RNA (up to 100 µl total RNA) by adding 0.1 volume 10× TURBO DNase buffer and 0.5 µl TURBO DNase (Thermo Fisher, AM1907), then incubating at 37 °C for 30 min. An additional 0.5 µl of TURBO DNase was then added followed by a second incubation. DNase was removed by adding 0.1 volume of DNase Inactivation Reagent. For each replicate, 40 µg of DNase-treated RNA was then used for eight reverse transcription reactions. For each reaction, 5 µg RNA was mixed with 1 µl 2 µM P7-pLSmp-assUMI-gfp and 1 µl of 10 mM dNTPs (NEB, N0447L), incubated at 65 °C for 5 min, and placed on ice. 6 µl of reverse transcription master mix was then added (4 µl 5× FS buffer, 1 µl 0.1 M dithiothreitol, and 1 µl SSIV (Thermo Fisher, 18091050)), and the reaction was incubated at 55 °C for 60 min, followed by 80 °C for 10 min. Each reverse transcription reaction was combined and split into 32 total PCR reactions per replicate. For PCR1, reactions contained 160 µl template, 640 µl 2× OneTaq master mix, 8 µl 100 µM indexed P5 primer (rand-P5-E##), 8 µl 100 µM P7 primer, and 464 µl water, and were split over 32 wells with the following cycling parameters: 2 min at 95 °C, followed by three cycles of 20 s at 95 °C, 30 s at 55 °C, and 2 min at 72 °C, with a final extension of 5 min at 72 °C, followed by a 4 °C hold. Reactions were pooled by replicate, cleaned up with AMPure XP beads at 1.8×, and eluted in 291 µl of 10 mM Tris pH 8.0. For PCR2, the entire 291 µl of eluate from PCR1 was used as input and combined with 300 µl OneTaq master mix, 3 µl 100× SYBR Green (Thermo Fisher, S7563), 3 µl 100 µM P5 primer, and 3 µl 100 µM P7 primer. This mix was split over 24 reactions for each replicate. Libraries were amplified and monitored in real-time by qPCR with 2 min at 95 °C, followed by cycles up to the qPCR inflection point consisting of 15 s at 95 °C, 20 s at 60 °C, and 2 min at 72 °C. Libraries were then pooled by replicate.  The three DNA libraries were pooled together, as were the three RNA libraries, and these two final pools were size-selected on 1% agarose gel for the expected band (156 bp). The RNA and DNA pooled libraries were quantified with Qubit dsDNA HS (Thermo Fisher) and diluted to 2 nM. Libraries were loaded at a ratio of 2:1 RNA:DNA and paired-end sequencing was performed on a NextSeq 2000 with the following primers and cycle numbers: read1 (BC forward): 16 cycles with primer TruSeq-R1; index1 (UMI): 10 cycles with primer randMPRAseq-I1; read2 (BC reverse): 16 cycles with primer randMPRAseq-R2; index2 (sample index): 10 cycles with primer TruSeq-I2.