‘Read–through marking’ reveals differential nucleotide composition of read-through and truncated cDNAs in iCLIP

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AI-generated summary by claude@2026-07, 2026-07-17

A modified iCLIP protocol with a 5' marker identified read-through cDNAs with adenosine-rich starts, confirming that truncated cDNAs accurately mark protein-RNA crosslink sites.

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AI-generated deep summary by claude@2026-07, 2026-07-17 · read from full text

This paper introduces “read-through marking” as an approach for analyzing iCLIP data, comparing nucleotide composition in read-through cDNAs versus truncated cDNAs to distinguish different sources or behaviors of cDNA ends. The work focuses on extracting sequence-compositional differences from iCLIP reads to inform interpretation of truncation and continuation patterns. A key caveat is that the analysis depends on assumptions about what read-through versus truncation represent in the specific iCLIP workflow and library preparation, so compositional signatures may vary with experimental context and processing. This paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

We established a modified iCLIP protocol, called ‘read-through marking’, which facilitates the detection of cDNAs that have not been truncated upon encountering the RNA–peptide complex during reverse transcription (read-through cDNAs). A large proportion of these cDNAs would be undesirable in an iCLIP library, as it could affect the resolution of the method. To this end, we added an oligonucleotide to the 5’-end of RNA fragments—a 5’-marker—to mark the read-through cDNAs. By applying this modified iCLIP protocol to PTBP1 and eIF4A3, we found that the start sites of read-through cDNAs are enriched in adenosines, while the remaining cDNAs have a markedly different sequence content at their starts, preferentially containing thymidines. This finding in turn indicates that most of the reads in our iCLIP libraries are a product of truncation with valuable information regarding the proteins’ RNA-binding sites. Thus, cDNA start sites confidently identify a protein’s RNA-crosslink sites and we can account for the impact of read-through cDNAs by commonly adding a 5’-marker.
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