A High-Throughput 3'-Tag RNA Sequencing for Large-Scale Time-Series Transcriptome Studies.

RNA sequencing (RNA-seq) has proven invaluable for exploring gene expression variation under complex environmental cues. However, the cost of standard RNA-seq (e.g., Illumina TruSeq or NEBNext) remains a barrier for high-throughput applications. 3'-Tag RNA-seq (3'-TagSeq) is a cost-effective solution that permits large-scale experiments. Unlike standard RNA-seq, which generates sequencing libraries for full-length mRNAs, 3'-TagSeq only generates a single fragment from the 3' end of each transcript (a tag read) and quantifies gene expression by tag abundance. Consequently, 3'-TagSeq requires lower sequencing depth (~5 million reads per sample) than standard RNA-seq (~30 million reads per sample), which reduces costs and allows increased technical and biological replication in experiments. Because 3'-TagSeq is considerably cheaper than standard RNA-seq while exhibiting comparable accuracy and reproducibility, researchers focusing on gene expression levels in large or extensive time-series experiments might find 3'-TagSeq to be superior to standard RNA-seq. In this chapter, we describe 3'-TagSeq sequencing library preparation and provide example bioinformatics and statistical analyses of gene expression data.