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Cell Biology JoVE (Open Access) Citable · DOI

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

DOI: 10.3791/59805-v
What you'll learn
  • ✓ Map transcription start sites at single-nucleotide resolution using CAGE methodology
  • ✓ Perform cap-trapping to enrich 5' capped mRNA molecules from low-input RNA samples
  • ✓ Generate high-quality CAGE libraries from nanogram quantities of total RNA
  • ✓ Validate library quality and assess RNA degradation levels before sequencing
Protocol

Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5’ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

Difficulty
advanced
Total time
~6–8 hours per sample (excluding sequencing)
Biosafety
BSL-1

Steps

1
Prepare DNA template and oligonucleotide components

Set up the DNA template and linker oligonucleotides required for reverse transcription and cap-trapping steps.

▶ 01:04
2
Perform reverse transcription of capped mRNA

Synthesize complementary DNA (cDNA) from mRNA using reverse transcriptase, generating cDNA:RNA hybrid molecules.

▶ 01:38
3
Purify cDNA:RNA hybrids using magnetic beads

Isolate cDNA:RNA hybrid molecules from the reaction using magnetic bead-based separation to remove unincorporated primers and enzymes.

▶ 02:24
4
Enrich 5' capped molecules through cap-trapping

Selectively capture RNA polymerase II transcription start sites by binding 5' cap structures, removing non-capped molecules from the library.

▶ 03:42
5
Assess RNA degradation and determine PCR cycle number

Evaluate RNA integrity and optimize PCR amplification cycles to ensure library quality without over-amplification.

▶ 05:16
6
Validate SLIC-CAGE library quality and composition

Confirm DNA quality, library insert size, and proper enrichment of capped 5' ends using analytical techniques.

▶ 05:39
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