Challenges and considerations for reproducibility of STARR-seq assays

STARR-seq, a popular assay for directly quantifying activity of thousands of enhancer sequences simultaneously, has seen limited standardization across studies. Further, the assay is long with >250 steps, and frequent customization of the protocol and variations in bioinformatics methods raise concerns for reproducibility of STARR-seq studies. Here, we assess each step of the protocol and analysis pipelines from published sources and in-house assays, and identify critical steps and quality control checkpoints necessary for reproducibility of the assay.