Step-by-Step Protocol for In Situ Profiling of RNA Subcellular Localization Using TATA-seq.

Area of Science:

• Cell Biology
• Molecular Biology
• Genomics

Background:

• Membrane-less organelles are crucial for cellular processes, regulating biomolecules via phase separation.
• Detecting RNA distribution within these dynamic hubs is essential but challenging.
• Existing methods lack efficiency and specificity for RNA profiling in these compartments.

Purpose of the Study:

• To present a novel, efficient protocol for in situ RNA profiling within membrane-less organelles.
• To enable detailed analysis of RNA subcellular localization and diversity.
• To provide a versatile tool for spatial transcriptomics.

Main Methods:

• Developed Target Transcript Amplification and Sequencing (TATA-seq) for in situ RNA detection.
• Utilized antibody-mediated targeting and T7 promoter-driven reverse transcription.
• Incorporated IgG control for background subtraction and T7 RNA polymerase for amplification.

Main Results:

• Achieved high-fidelity RNA amplification from low-input samples (<10,000 cells).
• Demonstrated optimized sequencing metrics with <25% duplication rate and ~90% mapping ratio.
• Validated TATA-seq's modularity for diverse organelles, including nuclear speckles and paraspeckles.

Conclusions:

• TATA-seq offers subcellular spatial resolution for RNA analysis in membrane-less organelles.
• The protocol ensures high sensitivity and yield, adaptable to various biological conditions.
• This method advances spatial transcriptomics and the study of RNA biology in cellular compartments.