Nov 6, 2018

High-throughput RNA-Seq analysis on plant response under fluctuating temperature conditions

BioRxiv : the Preprint Server for Biology
Mari KamitaniAtsushi J Nagano

Abstract

RNA-Seq has been contributed to understand biological phenomena as a method to measure whole transcriptome. However, being still costly and laborious, experiments in larger scales has been prevented. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method, which did not require enrichment step of mRNA and pooled all samples after reverse transcription (RT) step of library preparation. We added sample index sequences to each sample at RT step and pooled all samples into a single tube for the following steps. To suppress RT from A-rich sequences in rRNA, we conducted RT at higher temperature. We enabled single-read sequencing of libraries in addition to paired-end one. We found that the pooled RT products was included large amount of remained RNA, mainly rRNA, which caused over-estimation of cDNA quantity. The over estimation of cDNA resulted in unstable result of tagmentation, because the size distribution of final library was largely affected by input-amount of cDNA in tagmentation reaction. Therefore, degradation of RNA before tagmentation step was turned out to be necessary for the stable preparation of libraries. We applied our RNA-Seq method to analyse temperature responses in A...Continue Reading

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Mentioned in this Paper

Study
Size
Genes
Sequence Determinations, RNA
Radiosurgery
Ribosomal RNA
Rotational Test
Reverse Transcriptase Polymerase Chain Reaction
Sequencing
High Throughput Analysis

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