Studying enhancer activity by FAIRE-STARR-seq in the U2OS-GR and the U2OS-AR cells upon hormone treatment

To quantify functional enhancers, we performed STARR-seq (Self-Transcribing Active Regulatory Region sequencing) in the U2OS-GR and the U2OS-AR cell lines (derived from U2OS ATTC:HTB-96, stably transfected with an expression construct for rat GR or human AR, respectively). U2OS-GR cells were treated with dexamethasone (1 µM) or vehicle (ethanol) for 14 hours. U2OS-AR cells were treated with R1881 (5 nM) or vehicle (DMSO) for 14 hours. To limit the number of putative enhancers, the STARR library contained genomic regions isolated by FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements) from dexamethasone-treated U2OS-GR cells to include regions that gain accessibility upon GR activation. We added unique molecular identifiers (UMIs) during the reverse transcription stage to facilitate quantitative measurements of enhancer activity for each fragment. The UMI for each read is present within the sequence identifier line (directly following the y coordinate and separated by a ':') of the fastq files.