Dataset Information

Construction of High-Quality Rice Ribosome Footprint Library.

ABSTRACT: High-throughput sequencing of ribosome footprints precisely maps and quantifies in vivo mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library with peak reads of approximately 28-nucleotide (nt) length and strong 3-nt periodicity is constructed via key steps including rRNA depletion, end repair, 3' adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification. Biological significance of rice ribosome footprint library is further revealed by the comparison of transcriptomic and translatomic responses to salt stress and the utilization for novel open reading frame (ORF) identification. This improved protocol for rice ribosome footprint library construction will facilitate the global comprehension and quantitative measurement of dynamic translation in rice.

SUBMITTER: Yang X

PROVIDER: S-EPMC7500414 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Construction of High-Quality Rice Ribosome Footprint Library.

Yang Xiaoyu X Cui Jie J Song Bo B Yu Yu Y Mo Beixin B Liu Lin L

Frontiers in plant science 20200904

High-throughput sequencing of ribosome footprints precisely maps and quantifies <i>in vivo</i> mRNA translation. The ribosome footprint sequencing has undergone continuing development since its original report. Here we provide a detailed protocol for construction of high-quality ribosome footprint library of rice. Rice total polysomes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by co ...[more]

PMID: 33013996

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Project description:We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Dataset Information

Construction of High-Quality Rice Ribosome Footprint Library.

Publications

Construction of High-Quality Rice Ribosome Footprint Library.

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