Simultaneous Profiling of Transcripts and Genomic Regions Associated with Nuclear Bodies Using the RNA-DNA High-Salt...

Area of Science:

• Cell Biology
• Molecular Biology
• Genomics

Background:

• Nuclear functions like transcription and splicing depend on nuclear bodies, which are membrane-less organelles.
• These bodies assemble via phase separation and possess liquid-like properties, complicating compositional analysis.
• Understanding nuclear body composition is key to deciphering gene regulation in eukaryotic cells.

Purpose of the Study:

• To present a detailed protocol for the RNA-DNA High-salt Recovered Sequence (RD-HRS) method.
• To enable simultaneous profiling of RNA and genomic DNA within nuclear bodies.
• To overcome challenges in analyzing the composition of liquid-like nuclear microenvironments.

Main Methods:

• The study details the RNA-DNA High-salt Recovered Sequence (RD-HRS) method, an extension of HRS-seq.
• High-salt treatments are employed to render nuclear bodies insoluble.
• This facilitates the recovery and analysis of both RNA and DNA from the same sample.

Main Results:

• The RD-HRS method allows for the simultaneous profiling of RNA and genomic DNA content within nuclear bodies.
• This technique provides insights into the molecular composition of these crowded nuclear microenvironments.
• The protocol enables a more comprehensive understanding of nuclear body constituents.

Conclusions:

• The RD-HRS method offers a robust approach for characterizing the dual RNA and DNA composition of nuclear bodies.
• This technique advances the study of nuclear organization and its role in gene regulation.
• The RD-HRS method provides a valuable tool for investigating the molecular makeup of phase-separated nuclear compartments.