GSM7062374: HEK SCZ MPRA Rep 2; Homo sapiens; OTHER
RNA was extracted from each well using Qiagen RNeasy kit (Qiagen, cat#74004), using 10 uL of -Mercaptoethanol (Sigma-Aldrich, cat#60-24-2) per 1 mL of Qiagen RLT buffer. The columns were treated with DNase (Qiagen, cat#79256). To acquire an initial input of DNA put into the cells, DNA was extracted from the AAV2 virus which contained the AAV-MPRA library using a NucleoSpin virus kit (Macherey-Nagel, cat#740983.50). The AAV-variant-minP-GFP-barcode library was grown in 2 L of LB with ampicillin, and maxi prepped using Qiagen Maxi prep kit.  The UNC vector core packaged the AAV-variant-minP-GFP-barcode library into AAV serotype 2 (AAV2). The resulting virus had the titer of 7x1012 viral particles/uL. After cellular transfection and RNA extraction, cDNA was generated from the extracted RNA by SuperScript IV Reverse Transcriptase (Invitrogen, cat#18090050) using a primer that targets downstream of the barcodes. DNA extracted from the AAV-MPRA library and cDNA from each transduced well were amplified via PCR using NEBNext 2X Q5 Hifi HS Mastermix. The samples were cleaned up using Zymo clean and concentrator-5. This was followed by the second amplification step to add on sequencing adaptors and unique Illumina indices. Again, NEBNext 2X Q5 Hifi HS Mastermix was used for amplification. The resulting libraries were cleaned up using 0.75X ampure beads (Beckman Coulter, cat#A63881) and sequenced by UNC HTSF via Novaseq 6000 SP (1x35bp), with custom primers that capture the barcode sequence and sequencing index.