Dataset Information

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

ABSTRACT: Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

SUBMITTER: Puchta M

PROVIDER: S-EPMC7650657 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

Puchta Marta M Boczkowska Maja M Groszyk Jolanta J

Genes 20201013 10

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low R ...[more]

PMID: 33066221

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Project description:The emergence of NextGen sequencing technology has generated much interest in the exploration of transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at a much lower cost. We have developed a low-cost and robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries by combining several recent improvements. First, we designed balanced adapter sequences for multiplexing of samples; second, dUTP incorporation in 2(nd) strand synthesis was used to enforce strand-specificity; third, we simplified RNA purification, fragmentation and library size-selection steps thus drastically reducing the time and increasing throughput of library construction; fourth, we included an RNA spike-in control for validation and normalization purposes. To streamline informatics analysis for the community, we established a pipeline within the iPlant Collaborative. These scripts are easily customized to meet specific research needs and improve on existing informatics and statistical treatments of RNA-seq data. In particular, we apply significance tests for determining differential gene expression and intron retention events. To demonstrate the potential of both the library-construction protocol and data-analysis pipeline, we characterized the transcriptome of the rice leaf. Our data supports novel gene models and can be used to improve current rice genome annotation. Additionally, using the rice transcriptome data, we compared different methods of calculating gene expression and discuss the advantages of a strand-specific approach to detect bona-fide anti-sense transcripts and to detect intron retention events. Our results demonstrate the potential of this low cost and robust method for RNA-seq library construction and data analysis.

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Dataset Information

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

Publications

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

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