Functional definition of thyroid hormone response elements based on a synthetic Starr-seq screen

We performed an original protocol called synthetic STARR-seq (PMID: 30427832) in which a library of putative response elements are tested for their capacity to bind thyroid hormone (T3) nuclear receptors and convey T3 transactivation More than 10 000 putative T3 response elements (T3RE) were cloned in the hSTARR-seq-ORI vector with a minimal promoter, in the 3' end of the transcribed sequence. These are variant of the consensus T3RE (5'NGGTCANNNNRGGNNA3') Therefore the most active T3RE are over-represented in the RNA of cells transfected with the plasmid library and treated with T3. Overall design: After transfection of the STARR-seq plasmid library (600 g or 1200 ng, +/-T3) RNA was extracted, and the 3'end of the transcript containing T3RE was amplified by RT-PCR. Deep sequencing was used to address the influence of T3 treatment on the representation of individual T3RE. Counts table can be used to compair the frequency of a given T3RE is the input library, transfected cells without T3, and transfected cells with T3.