RAG-seq: NSR-primed and Transposase Tagmentation-mediated Strand-specific Total RNA Sequencing in Single Cells.

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Single-cell RNA sequencing (scRNA-seq) offers high resolution of cellular diversity.
• Existing scRNA-seq methods often struggle with full-length transcript capture and strand orientation.
• Accurate transcript quantification and antisense RNA detection require strand-specific information.

Purpose of the Study:

• To introduce RAG-seq, a novel strand-specific total RNA sequencing method.
• To overcome limitations of current methods in transcript coverage and strand information.
• To enhance sensitivity and reproducibility for low-input and single-cell samples.

Main Methods:

• RAG-seq utilizes not-so-random (NSR) primers and Tn5 transposase-mediated tagmentation.
• Optimized reverse transcription with oligo-dT primers is employed.
• Depletion of Abundant Sequences by Hybridization (DASH) removes rRNA, followed by linear amplification.

Main Results:

• RAG-seq provides comprehensive transcript coverage and maintains strand orientation.
• The method demonstrates enhanced sensitivity and reproducibility, particularly for single cells.
• Application in mouse oocytes and embryos identified stage-specific antisense transcripts.

Conclusions:

• RAG-seq represents a significant advancement in transcriptome analysis.
• The technique enables accurate quantification of overlapping genes and detection of antisense transcripts.
• RAG-seq offers valuable insights into the regulatory roles of antisense RNAs in early development.