Cleaner libraries: uses P-3′App-DNA adapters and a DBO during ligation to reduce side products/adapter dimers and loss of RNA. Validated on limited samples: the study highlights successful mapping from 5k cells (mouse hematopoietic progenitors and neurons).
What you measure/learn Active transcription dynamics (initiation, pausing, and elongation) rather than steady-state RNA levels—useful for mechanism and rapid responses
What they used it for (initial studies) Two Molecular Cell papers from the Sylvester team applied rPRO-seq to the Integrator complex: In neurons, INTS11 emerged as a regulator of genes implicated in neurodevelopmental disorders.
In stem-cell models, Integrator/INTS11 acts very early in development (as early as embryonic day 2) and likely at transcription initiation, revising prior views.
Practical notes & limitations It’s not single-cell; think low-input bulk. Still requires careful nuclei prep and run-on handling; results are complementary to, not a replacement for, steady-state RNA-seq (you’ll often want both).
For more on RNAseq and scRNA-seq, follow Rhymes with Haystack albertvilella.substack.com