PCRD-seq: proximity crosslinking-induced RNA depletion sequencing for low-input chromatin-associated transcriptome profiling.

Chromatin-associated RNAs (caRNAs) play critical roles in regulating chromatin organization and transcription, underscoring the importance of their study. Proximity labeling has emerged as a promising and versatile technique for profiling caRNAs with high spatiotemporal resolution. While being a powerful technique, traditional proximity labeling methods depend on complex, high-input enrichment protocols, which significantly limit their wide practical application. Here, we developed a straightforward, enrichment-free caRNA profiling strategy: proximity crosslinking-induced RNA depletion sequencing (PCRD-seq). This approach leverages the proximity crosslinking between chromatin and its surrounding RNAs induced by singlet oxygen generated by HoeDBF, a photosensitizer targeting chromatin region. The proximity crosslinking hinders the release of caRNAs during routine TRIzol extraction, consequently leading to a specific depletion of these RNAs. This method was successfully applied to investigate the role of U1 small nuclear RNA (snRNA) in RNA chromatin retention and the differences in chromatin-associated transcriptomes between two ovarian cancer cell lines with opposite metastatic capability. Moreover, our PCRD-seq exhibits potential in profiling nuclear lamina-associated RNAs, which paves the way for its application to profile RNAs associated with other chromatin subdomains. The minimal cell input and simple workflow render PCRD-seq a transformative tool for wide applications.