Amplifying STARR-Seq Transcripts from Transduced Cells

Total RNA was purified from transduced cells (Qiagen #75144) and concentrated 7.5X using an RNA Cleanup Kit (NEB #T2040L). RNA was reverse transcribed according to manufacturer guidelines with STARR-Seq transcript–specific primers (Supplemental Table 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206) and amplified using custom STARR-Seq transcript–specific primers (Supplemental Table 1) as previously described [15 (link)]. Diluted Lenti-STARR-Seq plasmid ‘input’ control was amplified using an input concentration that 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-150 bp sequencing (Novogene).