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Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

Mari Kamitani,Makoto Kashima,Ayumi Tezuka,A. Nagano

Published 2018 in bioRxiv

ABSTRACT

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions; its use in large-scale experiments is limited by costs and labour. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method that did not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62°C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, and caused over-estimations of the quantity of DNA, resulting in unstable tagmentation results. Degradation of RNA before tagmentation was necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq), and used it to investigate temperature responses in Arabidopsis thaliana. We analysed how sub-ambient temperatures (10–30°C) affected the plant transcriptomes, using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.

PUBLICATION RECORD

  • Publication year

    2018

  • Venue

    bioRxiv

  • Publication date

    2018-11-06

  • Fields of study

    Biology, Environmental Science

  • Identifiers
    DOI 10.1101/463596
  • External record

    Open on Semantic Scholar

  • Source metadata

    Semantic Scholar

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