Enhancing Transcriptome Mapping with Rapid PRO-seq Profiling of Nascent RNA.

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Precision nuclear run-on (PRO) sequencing (PRO-seq) maps polymerase activity and nascent transcripts.
• Current PRO-seq protocols are lengthy, complex, and require substantial starting material.

Purpose of the Study:

• To develop a faster, more efficient PRO-seq protocol (rPRO-seq).
• To overcome limitations of existing PRO-seq methods regarding time, technical difficulty, and sample input.

Main Methods:

• Developed rapid PRO-seq (rPRO-seq) using pre-adenylated single-stranded DNAs (AppDNA) and a dimer blocking oligonucleotide (DBO).
• Incorporated on-bead 5' RNA end repair and column-based purification.
• rPRO-seq enables transcriptome mapping within approximately 12 hours.

Main Results:

• rPRO-seq significantly reduces protocol time and increases ligation efficiency.
• Adapter dimers are abolished, and sample loss/RNA degradation are minimized.
• rPRO-seq demonstrates reproducibility comparable to standard PRO-seq and is scalable to 25,000 cells.

Conclusions:

• rPRO-seq is a robust, efficient, and scalable method for nascent transcript analysis.
• Applied rPRO-seq to identify *Ints11*'s role in mouse hematopoietic stem and progenitor cell homeostasis.
• rPRO-seq facilitates patient-specific genome-wide transcription profiling with minimal sample input.