TM3'seq: A Tagmentation-Mediated 3' Sequencing Approach for Improving Scalability of RNAseq Experiments

G3 (Bethesda). 2020 Jan 7;10(1):143-150. doi: 10.1534/g3.119.400821.

Abstract

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples -producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3'seq, a 3'-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3'seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human and Drosophila melanogaster RNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext kit. We expect that the cost- and time-efficient features of TM3'seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

Keywords: RNAseq; TM3seq; TagSeq; Transcriptomics; mRNA extraction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Costs and Cost Analysis
  • Drosophila melanogaster
  • Female
  • Humans
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA-Seq / economics
  • RNA-Seq / methods*
  • RNA-Seq / standards
  • Reproducibility of Results

Substances

  • 3' Untranslated Regions
  • RNA, Messenger