Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a key method for genome-wide RNA-binding protein mapping.
• HITS-CLIP is commonly employed to validate microRNA and other RNA-binding protein targets.
• Accurate identification of RNA-binding protein binding sites is crucial for understanding gene regulation.

Purpose of the Study:

• To address the issue of mispriming artifacts in HITS-CLIP data.
• To present a modified HITS-CLIP protocol that mitigates reverse transcription mispriming.
• To enhance the accuracy and sensitivity of binding site identification in HITS-CLIP experiments.

Main Methods:

• Implementation of a modified HITS-CLIP protocol.
• Analysis of HITS-CLIP libraries to identify and quantify mispriming artifacts.
• Comparison of standard and modified HITS-CLIP protocols for artifact reduction.

Main Results:

• Substantial mispriming during reverse transcription leads to overrepresented sequences complementary to the reverse transcription primer.
• Up to 45% of peaks in public HITS-CLIP libraries can be attributed to mispriming artifacts.
• Standard validation methods fail to distinguish artifactual peaks from true binding sites.

Conclusions:

• The developed modification effectively eliminates mispriming artifacts in HITS-CLIP.
• The improved protocol enhances the sensitivity and complexity of HITS-CLIP libraries.
• This advancement increases the reliability of genome-wide RNA-binding protein target identification.