Extended Data Figure 2. Transcription initiates within the core promoter of the STARR-seq construct.

5’ Rapid Amplification of cDNA Ends (5’ RACE) demonstrates that transcription initiates at the TCT and Initiator motifs within the hkCP and dCP, respectively. a, Setup of the 5’RACE experiment including the STARR-seq plasmid, used here with two defined enhancers, the STARR-seq transcript and location of all primers used to specifically amplify 5’-capped STARR-seq transcripts. b, 5’ RACE nested PCR products separated on a 1% agarose gel. c, Screenshot of Sanger sequencing results (chromatogram and called bases) compared with the template sequence. Annotations are shown in green, in the following order: 5’ RACE adapter, hkCP with TCT motif (only the part downstream of the transcription start is annotated, as the 5’ part is not present in the sequenced cDNA), spliced intron, GFP; the sequencing primer is shown in red (top). Also shown is a version that displays the template and Sanger sequencing results for the core promoter region only (zoom in). d, same as in (c) but for the dCP for which transcription initiates within the Initiator (Inr) motif.