web analytics

RNAmp: Ultra-Low-Input Library Construction Enabling Epitranscriptome Profiling From Scarce Samples

Lead Inventor: Chuan He

SUMMARY

More efficient profiling of RNA modifications, RNA–protein interactions, and translation from single-cell-scale samples is supported by an ultra-low-input RNA sequencing library method that prepares sequencing-ready libraries from immunoprecipitated RNA

The Unmet Need: Low-input RNA sequencing workflows can lose material and limit molecular profiling

  • Single-cell and other low-input sequencing applications are increasing demand for workflows that capture a broader range of RNA species while preserving scarce material. Methods that support direct profiling of RNA modifications, RNA–protein interactions, and translation could extend molecular analysis to samples that are difficult to study with conventional library preparation.

  • Existing approaches can depend on polyadenylated RNA or inefficient random priming, and may exhibit 3′-end bias. These limitations complicate sequencing of scarce or immunoprecipitated RNA.

The Proposed Solution: RNAmp prepares strand-specific sequencing libraries from ultra-low-input and immunoprecipitated RNA

  • The faculty inventor developed RNAmp- a direct next-generation sequencing library preparation workflow designed for RNA inputs below 100 pg, approximately the RNA content of a single cell, including RNA recovered by immunoprecipitation. The workflow fragments RNA, performs enrichment or immunoprecipitation, repairs RNA ends, adds poly(A) tails, and uses poly(dT)-primed, template-switching reverse transcription to generate cDNA. PCR adds a T7 promoter, followed by T7 polymerase linear amplification and a final reverse-transcription and PCR stage to produce strand-specific sequencing libraries. Carrier RNA reduces sample loss, while the approach avoids a separate ribosomal-RNA depletion step because rRNA is reduced in the final library; poly(A) tailing also supports capture of short fragments and naturally non-polyadenylated RNAs.

ADVANTAGES

  • Ultra-low-input RNA compatibility

  • At least 1,000-fold greater immunoprecipitated RNA efficiency

  • Reduced sample loss with carrier RNA

  • Strand-specific sequencing-ready libraries

  • Capture of short and non-polyadenylated RNAs

  • Reduced ribosomal RNA representation

APPLICATIONS

  • Single-cell m6A RNA modification profiling

  • RNA immunoprecipitation and cross-linking immunoprecipitation sequencing

  • Single-cell transcriptome and translatome analysis