STARR-seq library profiling 100 defined open chromatin regions (‘focused library’) in livers of male and female mice treated with the nuclear receptor CAR (Nr1i3) agonist ligand TCPOBOP.

Massively parallel reporter assays are widely used to discover functional enhancers but have largely been limited to transfected cell models, which are confounded by vector-induced innate immune responses and lack the physiologically relevant cellular and endogenous hormonal context and chromatin environment of complex mammalian tissues. Here, we combine hydrodynamic injection with a modified STARR-seq-based MPRA to determine condition-specific enhancer activity in mouse liver at scale. 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 promoter. We prepared a focused STARR-seq library comprised of 100 PCR-amplified open chromatin regions nearby genes showing sex-biased expression or responsiveness to TCPOBOP, a xenobiotic and agonist ligand of the nuclear receptor CAR (Nr1i3). We assayed STARR-seq activity for the 100 genomic regions in male liver, in female liver and in TCPOBOP-treated male liver to quantitatively measure their intrinsic transcriptional activity under the 3 indicated biological conditions, and thereby identified enhancers whose activity is sex-dependent or xenobiotic-responsive. The focused plasmid library was delivered to mouse liver by hydrodynamic tail vein injection (HDI). Three types of Illumina sequencing libraries were prepared and are included in this GEO submission: the original STARR-seq plasmid libraries used for HDI, STARR-seq DNA libraries extracted from the transfected livers, and STARR-seq RNA libraries prepared from STARR-seq reporter RNA extracted from each transfected liver. These libraries were all generated by PCR amplification carried out in parallel and under the same PCR conditions. The STARR-seq plasmid library is the same material used for HDI and indicates the relative abundance of each reporter sequence mapping to the 100 open chromatin (DNase I-hypersensitive; DHS) regions used for library preparation. The STARR-seq DNA plasmid library was prepared using DNA reporter-specific primers that amplify plasmid DNA extracted from the transfected livers. STARR-seq enhancer activity was calculated by dividing the Illumina sequencing read counts of each RNA reporter (numerator) by that of the corresponding STARR-seq plasmid or DNA library sequence reads (denominator). The STARR-seq RNA libraries were prepared from poly-adenylated RNA extracted from the HDI livers were reverse-transcribed followed by DNase treatment, and then amplified using RNA reporter-specific primers; these RNA libraries represent the self-transcribing activity of each DHS region included in the STARR-seq plasmid library.