(d) extraction and purification of DNA and RNA from the cells, and (e) quantitation of the relative amounts of RNA and DNA for each oligonucleotide by next-generation sequencing. We tested expression in two neuroblastoma cell lines, SH-SY5Y (CRl-2266, ATCC, Manassas, VA) and SK-N-BE(2) (CRL-2271, ATCC). Both cell lines were cultured in a 1:1 mixture of EMEM (302003, ATCC) and F12K medium (10025-CV, Thermo Fisher Scientific, Waltham, MA) with 10% (vol/vol) fetal bovine serum (302020, ATCC) and 1% penicillin and streptomycin. Six independent biological replicates were conducted for each cell line. At 42 h post transfection, both plasmid DNA and RNA were extracted from the cells using the miRNeasy mini kit (217004, Qiagen, Germantown, MD) per manufacturer’s instructions. The cDNAs were synthesized from total RNA using QuantiTech Reverse Transcription kit (205311, Qiagen) with primers complementary to a sequence in the reporter vector 3’ of the inserted sequence. The target sequences from the cDNAs and also from the plasmid DNA extracted from the same cells were amplified using two-step PCR with primers containing sample barcodes to separately tag cDNA (representing expression from an allele) and input DNA from each replicate, unique molecular indices (UMI), and partial Illumina sequencing adapters (Illumina, San Diego,
