Protocol for simultaneous profiling of transcription start sites and full-length transcripts from low-input samples...

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Cap analysis of gene expression (CAGE) identifies transcription start sites (TSS) and promoter activity.
• Existing methods like CAGE require substantial RNA input and cannot profile entire transcript bodies.
• There is a need for low-input methods capable of simultaneous full-length transcript and TSS profiling.

Purpose of the Study:

• To present Smart-seq+5', a novel low-input protocol for characterizing full-length transcripts.
• To enable simultaneous identification of precise transcription start site (TSS) locations.
• To improve upon existing transcriptomic techniques by increasing sensitivity and accommodating diverse RNA types.

Main Methods:

• RNA extraction with optional in vitro polyadenylation.
• Reverse transcription and 5' capture.
• Tn5 tagmentation for comprehensive transcript coverage.
• Computational analysis for data interpretation.

Main Results:

• Smart-seq+5' successfully characterizes full-length transcripts.
• Precise TSS locations are identified concurrently with transcript profiling.
• The protocol demonstrates improved sensitivity compared to standard methods.
• Applicable to both polyadenylated and non-polyadenylated RNA samples.

Conclusions:

• Smart-seq+5' is an effective low-input method for comprehensive transcriptomic analysis.
• This protocol advances the ability to study gene expression regulation at the TSS and transcript body level.
• Smart-seq+5' offers enhanced sensitivity and versatility for various RNA samples.