Global analysis of enhancer targets: Bulk RNA-Seq validation

Single-cell perturbation assays such as Mosaic-seq enable highly multiplexed functional assessment of enhancers in their endogenous genomic context. By introducing a few computational and experimental improvements, we expanded the Mosaic-seq analysis to capture the secondary gene targets of enhancers. Our analysis of >500 putative enhancers in K562 cells demonstrates that many secondary hits are shared among enhancers targeting different transcriptional factors, which reveals an interwoven enhancer-driven gene regulatory network. Together, our data underscore the flexibility of manipulating gene transcription by modifying enhancer activity. Overall design: In order to validate the differentiated expressed genes identified in the Mosaic-seq experiment, we performed bulk RNA-seq for two enhancer regions, including the enhancer of ARL15 gene (ARL15-enh) and the third enhancer for the MYB gene (MYB-enh-3). For sample preparation, K562-dCas9-KRAB cells were infected with all 10 sgRNAs from each region, respectively. In addition, two non-targeting sgRNAs were used as the negative control. For each sgRNA, two to three biological duplicates were collected at the same time.