BioProject

Massively parallel reporter assays (MPRA) 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. More...

Massively parallel reporter assays (MPRA) 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. Analysis of a global STARR-seq library, comprised of ~50,000 genomic sequences released by DNase-I digestion of mouse liver nuclei, identified condition-specific enhancers and revealed strong correlations between liver enhancer activity and the chromatin state of the corresponding endogenous genomic regions. Overall design: A global STARR-seq reporter library was prepared by cloning into a STARR-seq vector with a minimal Albumin promoter ~50,000 genomic regions released by limited DNase-I digestion of mouse liver nuclei. The global reporter 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 global STARR-seq plasmid libraries used for HDI, global STARR-seq DNA libraries extracted from the transfected livers, and global 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 ~50,000 genomic regions (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. Less...