Fig. 1

Lenti-STARR Approach. A Putative regulatory sequences enriched by ATAC-Seq are used as input into Lenti-STARR-Seq. ATAC-Seq was performed on total human CD4+ T cells from peripheral blood obtained from healthy adult donors. The ATAC-Seq library is cloned into a promoter-less STARR-Seq screening vector that uses a bacterial origin of replication as a cryptic promoter [25]. The screening library is packaged in an HIV-enveloped non-integrating lentivirus, and transduced into resting CD4+ T cells. After 24 h in culture, STARR-Seq RNA is collected, reverse-transcribed using a transcript-specific primer, PCR-amplified, and prepared for next-generation sequencing (NGS). Created with BioRender.com B The Lenti-STARR-Seq screening vector carries an ATAC insertion site downstream of chimeric intron. C Enhancers are called with MACS2 [-log10(P) > 75], comparing STARR-Seq–obtained RNA reads to input plasmid reads [29]. Negative regulatory elements (NREs) are called using MACS2 [-log10(P) > 30] comparing input plasmid to STARR-Seq-obtained RNA reads [29]. Mean tag density is displayed, compared to ‘All’ accessible peaks from input. Reads are centered at CRE peak center. D Pearson correlation of tags across input ATAC-Seq peaks, Enhancers, or NREs for each STARR-Seq human CD4+ donor (N = 4)