SERIPH: A Two-Step Extraction Protocol for Selective Enrichment of Semi-Extractable RNAs

Conventional RNA extraction with acid guanidinium thiocyanate-phenol-chloroform (AGPC) reagents incompletely recovers a subset of transcripts, termed semi-extractable RNAs (seRNAs). This underrepresented RNA population includes architectural RNAs (arcRNAs), which scaffold membraneless organelles, as well as stress-induced downstream-of-gene readthrough transcripts (DoGs). Although our previously developed "improved" AGPC protocol, which incorporates a stronger physical disruption step, enhances seRNA recovery, it also co-extracts abundant, readily extractable RNAs, resulting in mixed populations that hinder precise characterization. Here, we present SERIPH (Semi-Extractable RNA Isolation from the InterPHase), a simple two-step protocol that selectively enriches seRNAs by separating them from readily extractable RNA species. In SERIPH, the aqueous phase containing readily extractable RNAs is first removed following conventional AGPC extraction. The remaining interphase is subsequently subjected to the improved extraction procedure, yielding a fraction selectively enriched for semi-extractable transcripts. Transcriptome-wide RNA sequencing demonstrates that SERIPH robustly enriches established seRNAs, including arcRNAs and stress-induced DoGs, while efficiently depleting highly abundant mRNAs. Compared with the improved method alone, SERIPH increases both the number and genomic extent of detectable DoG loci and enhances sensitivity for weakly semi-extractable transcripts that fall below conventional threshold-based classifications. SERIPH further reveals enrichment of intron-retaining transcript subsets within the semi-extractable RNA fraction. By expanding the detectable seRNA repertoire and enabling selective enrichment, SERIPH establishes a practical framework for focused, high-resolution analysis of seRNA populations. This methodological advance facilitates more comprehensive characterization of seRNAs and advances studies of RNA processing, transcriptional regulation, and RNA-mediated nuclear organization in stress responses and disease.