Protocol for direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Cap analysis of gene expression (CAGE) is crucial for identifying RNA transcript start sites (TSS).
• Adapting CAGE to new sequencing technologies is essential for continued genomic research.

Purpose of the Study:

• To present a protocol for CAGE using Illumina patterned flow cell technology.
• To enable accurate TSS and enhancer determination on modern sequencing platforms.

Main Methods:

• Utilized Cap analysis of gene expression (CAGE) on Illumina patterned flow cell technology.
• Incorporated dual indexes for mouse and human samples.
• Developed automated data processing and analytical framework.

Main Results:

• Successfully applied CAGE to mouse and human samples using Illumina sequencing.
• Established a complete analytical framework for CAGE data.
• Ensured CAGE operability for TSS and enhancer determination.

Conclusions:

• The presented protocol enables CAGE on current Illumina platforms.
• This facilitates robust identification of TSS and enhancers.
• The method supports ongoing genomic and transcriptomic studies.