GSM7501315: HepG2 cells, RNA_Rep1; Homo sapiens; OTHER
We produced lentivirus in 293FT cells using established protocols and filtered media (0.45 uM) to obtain virus. We seeded HepG2 cells in 6-well plates (1 million cells per well), and 24 hours post-seeding spinfected virus into HepG2 cells using established protocols (2000 RPM, 1.5 hours). After 48 hours incubation, we extracted RNA and DNA from cells using Norgen Total RNA purification kit and Qiagen DNeasy Blood & Tissue kit, respectively; we purified three replicates, using 8 wells per replicate (16 RNA columns per replicate). Lentiviral MPRA data was produced by following the protocol in Gordon et al 2020 (PMID 32641802), with minor modifications. Oligos (n=138,420, Supplemental Table 6) were synthesized by Twist Bioscience, and were composed of 170 nucleotides test element with 15 nucleotides 5' flanking sequence and 15 nucleotides 3' flanking sequence (Supp Table X). First-round amplification of the oligo library was performed as described with 5BC-AG-f01 and 5BC-AG-r01, but a separate first-round amplification was performed in parallel with custom primers 5BC-AG-f01B (5'-CTCACTCAGCCTGCATTTCTGCCAGGGCCCGCTCTAGACCTGCAGGTCGGTTCACGCAATG-3') and 5BC-AG-r01B (5'- GCTTTCGCTTAGCGATGTGTTCACTTTGCACAGTACCGGATTGCCAAGCTGGAAGTCGAGCTTCCATTATATACCCTCTAGTGAGGACCGGATCAACT-3'). These two reactions gave one library with test elements in forward orientation and one library with test elements in reverse orientation. These libraries were combined in equal quantities before second-round PCR amplification. For barcode association sequencing, we modified the read 1 and read 2 sequencing primers for our dual orientation library to (read 1) 5'- CTGCATTTCTGCCAGGGCCCGCTCTAGACCTGCAGG-3' and (read 2) 5'- TGGAAGTCGAGCTTCCATTATATACCCTCTAGTG-3'. All other primers and protocol steps followed the published method. Lentiviral MPRA as described in PMID 32641802