Poly(A)-Seq

Poly(A)-seq is a next-generation sequencing method that allows for transcriptome-wide profiling of polyadenylation sites. By selectively sequencing the 3' ends of mRNA molecules that have poly(A) tails, our Poly(A)-seq service provides a comprehensive view of mRNA stability and translation efficiency.

Overview of Poly(A)-Seq

Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Dynamic poly(A) tail regulation in the nucleus and cytoplasm has been implicated in various cellular and physiological processes. The next-generation sequencing provides ample opportunity to precisely delineate poly(A) tails. Various methods for profiling transcriptome-wide poly(A) sites were developed. Poly(A)-seq is a direct poly(A) sequencing method at the whole genome level. Therefore, Poly (A) -seq can be used as a potential tool to analyze the dynamic regulation process of mRNA turnover, degradation and translation, and it can also discover some unknown characteristics of RNA cracking and tailing.

Fig1 Overview of the function of mRNA poly(A) tails as master regulators of gene expression in the cytoplasm. (Passmore, 2022)

Library Construction of Poly(A)-Seq

One of the unique features of Poly(A)-Seq library construction is its enrichment for mRNA. The poly(A) tails of mRNA molecules are specifically captured using oligo(dT) beads or other poly(A) binding methods, which allows for the analysis of gene expression and post-transcriptional regulation at the mRNA level. This results in higher specificity and sensitivity compared to other sequencing methods. Another speciality of Poly(A)-Seq library construction is its whole-genome coverage. It provides transcriptome-wide coverage of poly(A) sites, which allows for the detection of novel poly(A) sites that have not been previously annotated in reference genomes.

Project Workflow

Data analysis

• Poly (A) length distribution of transcripts
• Poly (A) length difference analysis at gene/transcript level between groups
• Correlation analysis between Poly (A) length and transcript expression
• Joint analysis of 3'UTR length and poly (A) length
• Joint analysis of alternative polyadenylation (APA) and Poly (A) length

Deliverable: FastQ, BAM, QC report, global profiling of poly(A), custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Alignment, classification, and functional annotation of all the mapped reads
• Statistical analysis – sequencing error rate distribution, length distribution, multi-parameter data analysis, expression analysis, comparisons of transcript copy number, etc.
• Differential expression analysis of transcripts/genes
• GO annotation
• KEGG analysis
• Detection and annotation of Indels/SNPs

Sample Requirements

• RNA sample: quantity ≥ 2 ug, concentration ≥ 200 ng/uL.
• 1.8 ≤ OD_260/280 ≤ 2.2, OD_260/230 ≥ 2.0, RIN ≥ 7.0, 28S:18S ≥ 1.0.
• Please make sure the RNA is not degraded and contaminated.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

1. Passmore, Lori A., et al. Roles of mRNA poly (A) tails in regulation of eukaryotic gene expression. Nature Reviews Molecular Cell Biology 23.2 (2022): 93-106.
2. Yu, Fengyun, et al. Poly (A)-seq: A method for direct sequencing and analysis of the transcriptomic poly (A)-tails. PloS one 15.6 (2020): e0234696.