Poly(A)-ClickSeq Library Prep Kit 12 Reactions

Poly(A)-ClickSeq (PACSeq) is a targeted RNA sequencing technique that focuses specifically on sequencing the 3′ ends of polyadenylated RNA, such as eukaryotic mRNAs. As a specialized form of RNA-Seq, PACSeq offers a streamlined and cost-effective approach for profiling poly(A) sites and alternative polyadenylation, while maintaining key advantages of traditional RNA-Seq methods.

Unlike conventional whole-transcript sequencing, PACSeq avoids unnecessary fragmentation and emphasizes the biologically important 3′ UTR region.

Comparison of RNAseq technique with PACseq technique.

The technology behind PACSeq:

PACSeq utilizes a modified reverse transcription reaction that includes three chain-terminating nucleotides next to the natural ones:

3′-Azido-2′,3′-dideoxy-ATP

3′-Azido-2′,3′-dideoxy-GTP

3′-Azido-2′,3′-dideoxy-CTP

No thymidine analog is used, so reverse transcription only is able to be terminated after the last adenine (A) of the poly(A) tail, ensuring selective capture of the 3′ UTR region. For faster chain termination a different ratio of natural to terminating azido nucleotides is used in PACseq in full sequence reads. This  new approach enables both poly(A) site mapping and gene expression profiling in one workflow.

Advantages of PACSeq Over Traditional RNA-Seq

• No fragmentation required
• No enzymatic ligation, reducing artifacts and recombination
• Works with fragmented or degraded RNA
• Stranded technique provides strand-of-origin information
• Ideal for rare recombination or chimeric event detection
• Low input required as little as 10 ng total RNA
• Rapid protocol, libraries ready in ~6 hours
• Unique Molecular Identifiers (UMIs) are available

PACSeq maintains the robust performance of RNA-Seq while simplifying the workflow, lowering costs, and enriching for biologically relevant 3′ ends.

Applications

• Mapping of polyadenylation sites
• Studies of alternative polyadenylation (APA)
• Gene expression profiling with 3′ bias
• Quantifying transcript isoforms and post-transcriptional regulation
• Suitable for degraded RNA samples (e.g. FFPE)

Kit Components and Requirements

The Poly(A)-ClickSeq Library Prep Kit includes all core reagents required for PACSeq library generation except PCR primers.

If needed, a set of 12 compatible i7 index primers can be purchased separately here. Alternatively, users may provide their own primers if already validated for Illumina sequencing workflows.

LITERATURE

For a detailed list of publications utilizing PACseq technology please visit:

https://www.clickseqtechnologies.com/resources/polya-clickseq-papers