Longjian Niu, Jing Wan, Jialei Sun, Yingzhang Huang, Na He et al.
STARR-seq assesses millions of fragments in parallel measuring enhancer activity quantitatively. Here we show that STARR-seq is critically flawed with a systematic error in the cells of Arabidopsis thaliana (A. thaliana). Large amount of self-transcripts (STs) is lost during reverse transcription because these STs are polyadenylated after alternative polyadenylation sites (APAS) inside the test sequences. We solved this problem by using specially designed primer and recovered self-transcribed sequences independent from the PAS usage. In A. thaliana, we identified active enhancers and also enhancers quiescent in their endogenous genomic loci. Different from traditional STARR-seq identified enhancers, enhancers identified by new method are highly enriched in sequences proximal to the 5’ and 3’ ends of genes, and their epigenetic states correlate with gene expression levels. Our solution applies to methods based on self-transcript quantification. In addition, our results provide an invaluable functional enhancer activity map and insights into the functional complexity of enhancers in A. thaliana.
Longjian Niu, Jing Wan, Jialei Sun, Yingzhang Huang, Na He, Li Li, Chunhui Hou. Resolving a Systematic Error in STARR-seq for Quantitative Enhancer Activity Mapping. 2020-10-21. https://doi.org/10.1101/2020.10.20.346908
The same genome-wide 500–800-bp Arabidopsis genomic-fragment library was assayed in leaf protoplasts with the improved iSTARR-seq reverse-transcription design. Merged reporter cDNA and plasmid DNA signal from two biological replicates quantify activity while recovering transcripts terminated at internal alternative polyadenylation sites.
A genome-wide library of 500–800-bp sonicated A. thaliana genomic fragments was cloned into a pPBI221-based self-transcribing reporter and transfected into leaf protoplasts. The experiment compares merged reporter cDNA and plasmid DNA signal from two biological replicates to quantify enhancer activity.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 5 files (ZIP)GSE157030_family.soft.gzGSE157030_iSTARR-seq_cDNA_merge.bwGSE157030_iSTARR-seq_plasmid_merge.bwGSE157030_STARR-seq_cDNA_merge.bwGSE157030_STARR-seq_plasmid_merge.bw