3'Pool-seq: an optimized cost-efficient and scalable method of whole-transcriptome gene expression profiling.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Next Generation Sequencing (NGS) offers high accuracy but is limited by the cost of full-length mRNA sequencing.
• Developing cost-effective and scalable RNA sequencing methods is crucial for broader accessibility.

Purpose of the Study:

• To introduce 3'Pool-seq, a novel, cost-effective, and scalable RNA sequencing method.
• To demonstrate the accuracy and efficiency of 3'Pool-seq for bulk RNA sequencing.

Main Methods:

• 3'Pool-seq focuses sequencing on the 3'-end of mRNA, drawing from SMART-seq, Drop-seq, and TruSeq methodologies.
• Optimization of input RNA concentrations, tagmentation conditions, and read depth was performed for bulk RNA.
• A dual indexing scheme was adapted for a 96-well plate format, utilizing ERCC spike-ins for quality control.

Main Results:

• 3'Pool-seq achieves accurate gene expression quantification (Pearson's r = 0.96) and differential gene detection (AUC = 0.921) compared to TruSeq.
• The protocol is streamlined, taking less than 12 hours, and reduces costs by over 90% without custom reagents.
• The method successfully distinguished molecular signatures of troglitazone and pioglitazone treatments in HepG2 cells.

Conclusions:

• 3'Pool-seq provides gene expression detection on par with TruSeq at a fraction of the cost.
• Its hybrid indexing and streamlined workflow are adaptable to various formats, including 96-well plates, enabling multi-condition studies.
• Careful experimental design is necessary to account for potential pooling effects, with ERCC spike-ins aiding in analysis.