The advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the high costs of full-length mRNA sequencing have posed a limit on the accessibility and scalability of the technology. To address this, Pfizer researchers have developed 3’Pool-seq: a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3′-end of mRNA. The researchers drew from aspects of SMART-seq, Drop-seq, and TruSeq to implement an easy workflow, and optimized parameters such as input RNA concentrations, tagmentation conditions, and read depth specifically for bulk-RNA.
Thorough optimization resulted in a protocol that takes less than 12 h to perform, does not require custom sequencing primers or instrumentation, and cuts over 90% of the costs associated with TruSeq, while still achieving accurate gene expression quantification (Pearson’s correlation coefficient with ERCC theoretical concentration r = 0.96) and differential gene detection (ROC analysis of 3’Pool-seq compared to TruSeq AUC = 0.921). The 3’Pool-seq dual indexing scheme was further adapted for a 96-well plate format, and ERCC spike-ins were used to correct for potential row or column pooling effects. Transcriptional profiling of troglitazone and pioglitazone treatments at multiple doses and time points in HepG2 cells was then used to show how 3’Pool-seq could distinguish the two molecules based on their molecular signatures.
A schematic representation of the 3’Pool-seq protocol
The use of anchored oligo-dT primers with standard indexed TruSeq i7 adapter overhangs for first strand synthesis allows immediate pooling of multiple samples after reverse transcription. Within a pool, each sample can be uniquely identified by the TruSeq i7 index. Once pooled, purification, PCR, and Nextera tagmentation reagents are used to generate cDNA fragments. A second PCR step using standard TruSeq i7 and indexed Nextera i5 adapters allows selective amplification of only 3′-end cDNA fragments and barcoding of each sample pool with a standard Nextera i5 index. The final product is a dual-indexed hybrid Nextera/TruSeq 3′-library where the i5 Nextera index serves as the pool index, and the i7 TruSeq index serves as the sample index within a pool. Multiple indexed library pools can be further quantified and combined in equal proportions into a superpool for sequencing
3’Pool-seq can accurately detect gene expression at a level that is on par with TruSeq, at one tenth of the total cost. Furthermore, its unprecedented TruSeq/Nextera hybrid indexing scheme and streamlined workflow can be applied in several different formats, including 96-well plates, which allows users to thoroughly evaluate biological systems under several conditions and timepoints. Care must be taken regarding experimental design and plate layout such that potential pooling effects can be accounted for and corrected. Lastly, further studies using multiple sets of ERCC spike-ins may be used to simulate differential gene expression in a system with known ground-state values.