STARR-seq of artifical enhancer seqeunces consisting of homotypic and heterotypic TF binding site repeats

STARR-seq libraries of artificial enhancer sequences created by combining one or two transcription factor (TF) motifs in various combinations were used to assess cooperativity between TF motif pairs. We designed STARR-seq libraries that contained combinations of one or two different motifs with varying motif numbers (1 to 5) and using two different spacer lengths between motif instances (5bp or 10bp). A total of 8 different TF motifs were included and three different seqeunce contexts were used as flanking seqeunces for each motif combination. 12bp random barcodes were added to the insert libraries by PCR before cloning the fragments into the STARR-seq vector. We designed STARR-seq libraries that contained combinations of one or two different motifs with varying motif numbers (1 to 5) and using two different spacer lengths between motif instances (5bp or 10bp). A total of 8 different TF motifs was included and three different seqeunce contexts were used as flanking seqeunces for each motif combination. 5bp spacer libraries were also stably integrated into MEC1 cells using CRE-recombinase-mediated cassette exchange as part of an endogenous version of the STARR-seq assay.