STARR-seq enhancer activity determined following hydrodynamic delivery of STARR-seq plasmid library to mouse liver using three different STARR-seq promoters: Super Core promoter, ORI promoter, and minimal Albumin promoter [G173]

Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale, and we examine how different promoters affect STARR-seq reporter activity. Strong liver enhancer activity was observed with STARR-seq libraries containing an Albumin minimal promoter but not when using a Super Core promoter or an origin of replication (ORI) promoter. This work is part of a larger study where we prepare a global STARR-seq library, comprised of ~50,000 genomic sequences released by DNase-I digestion of mouse liver nuclei, and where we identify condition-specific enhancers with strong correlations between liver enhancer activity and the chromatin state of the corresponding endogenous genomic regions. Genomic DNA fragments released by DNase I digestion of mouse liver nuclei were pooled and cloned into STARR-seq reporter plasmids. Three such global-scale STARR-seq libraries were prepared (designated STARR-TYC4, STARR-TYC5 and STARR-TYC6), differing only in the promoter sequences used to drive reporter activity: Alb, corresponding to a minimal Alb promoter, and Super Core-1 (SCP1) and Origin (ORI) promoters, both commonly used in STARR-seq assays. Each library was delivered to mouse liver by hydrodynamic injection, followed by extraction of plasmid DNA and reporter RNA; extractions were carried out in parallel, using the same amount of starting material from each liver. Although the recovery of plasmid DNA was similar for all 3 promoters constructs, the yields of PCR-amplified RNA libraries from the ORI-STARR-seq and SCP1-STARR-seq libraries were 5-10-fold lower than for the pPromALB-STARR-seq library. Furthermore, after Illumina sequencing (Fastq files in this GEO record), the mapped RNA reads were 50 to 150-fold lower for the SCP1-STARR-seq and ORI-STARR-seq RNA libraries (mean mapped reads = 0.57 and 1.87 million, respectively) as compared to the pPromALB-STARR-seq RNA libraries (89.5 million mapped fragments); c.f., 41-56 million mapped recovered DNA sequence reads. Thus, the Super Core-1 and ORI promoters do not support HDI-STARR-seq reporter activity in mouse liver.