GSM7073577: hCD4 STARR Donor III _2; Homo sapiens; OTHER
HEK-293T Cells were grown in 15cm2 dishes in DMEM (Gibco #11995-065) with 10% FBS (R&D Systems #S11150). Cells were grown to 70% confluency, then DMEM was replaced two hours before transfection. Each 15cm2 plate is transfected with a total of 21ug plasmid DNA (1:2:1 ratio psPAX2-D64V:pATAC-STARR:pLAI-Env) [psPAX-D64V (Addgene #63586), and pLAI-HIV (Addgene #133996)]. DNA was mixed with 4.25x PEI (Polysciences #23966) in at total of 2mL DPBS per plate, let rest for complex formation for 10 minutes at 22°C, then added dropwise to the cell culture while swirling. 10 hours after transfection the  cells were washed once with DPBS, then cultured in DMEM with 10% FBS and 2mM Caffeine (Millipore #205548). 48 hours after transfection the media was collected and stored at 4°C. 72 hours after transfection the media is collected and pooled. The viral supernatant was sterile filtered through a 0.45uM PES filter, and loaded into Ultracentrifuge Tubes (Beckman #344058), and spun for 2.5 hours, 4°C, at 35,000g. Each pellet was dissolved in 200uL DPBS, which was left overnight at 4°C to resuspend. Concentrated Lentivirus was filtered through 0.45uM PES filter, and snap frozen in an ethanol/dry-ice bath at -80°C until use. Lenti-STARR Transduction: Freshly isolated hCD4 T Cells were suspended to 6.6E6 cells per mL in CTS Optimizer, then combined with 0.33mL concentrated lentivirus per mL cells. Cells were spinfected in low-bind 6-well plates (Corning #3471) with 7ug/mL polybrene (MilliporeSigma #TR1003G) at 950g, 32°C, for 2 hours. Immediately following spinfection, cells were gently resuspended and cultured for 2 hours at 37°C, 5% CO2. Cells were collected and centrifuged and suspended cultured for 24 hours. Cells are treated with 100U DNaseI (NEB M0303S) for 30 minutes at 37°C. Total RNA was purified from transduced cells (Qiagen #75144) and concentrated 7.5x using RNA Cleanup (NEB #T2040L). RNA was Reverse Transcribed according to manufacturer guidelines for STARR-Seq transcript specific primers (Supplemental File 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206). cDNA was amplified using a custom STARR-Seq transcript specific primer (Supplemental File 1) as previously described.2 Diluted Lenti-STARR-Seq plasmid 'input' control was amplified using an input concentration which yielded the same amplification cycle number as the STARR library for hCD4+ Donor I. PCR amplified library was purified using PCR cleanup columns (Qiagen #28206), and quantified for PE-150bp sequencing (Novogene).