An improved method of constructing degradome library suitable for Illumina high-throughput sequencing platform
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ABSTRACT:
PROVIDER: PRJNA576350 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10242902.fastq.gz | Fastqsanger.gz |
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Project description:Post-transcriptional gene regulation is a critical layer of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The miRNA-guided cleavage on the mRNA targets can be confirmed by analyzing the sequenced degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries is not as straightforward as sequencing small RNA libraries. Moreover, the currently used degradome or PARE methods utilize Mme1 restriction site and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the family members of the same gene family. In this modified degradome protocol, EcoP15I recognition site is introduced to the 3' end of the 5’RNA adaptor of TruSeq small RNA library, the double strand DNA adaptor sequence is modified to suit with the ends generated by the EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation. Therefore, degradome library generated using this protocol can be sequenced as easily as small RNA library, and the resulting tag length is ~27-nt, which is longer than previous methods (20-nt). The protocol allows sequencing small RNA and degradome libraries simultaneously.
2019-12-17 | GSE138545 | GEO
Project description:BackgroundPost-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5' RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal.ResultsIn this protocol, 3' end of the 5'RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library.ConclusionsDegradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.
| S-EPMC6859640 | biostudies-literature
Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean. one sample, We deep sequenced a degradome library constructed from different soybean tissues.
2011-11-02 | E-GEOD-33379 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE33378: Deep sequencing of small RNAs from different tissues in soybean GSE33379: Deep sequencing of the degradome cDNA library in soybean Refer to individual Series
2011-11-02 | E-GEOD-33380 | biostudies-arrayexpress
Project description:To identity the targets of miRNAs, we bundled 12 samples from different developing satages into four mixture samples. These samples were used to cosntruct degradome libraries and preform degradome sequencing on Illumina Hi-seq 2000 analyzer. More than 44.98 millions clean reads were obtained and 33.52 million reads were mapped to the soybean cDNA. The mapped reads were used to identity miRNA targets by CleaveLand4 pipeline. 4 degradome mixed samples, no replicates, but every degradome data consists of two parts data. Please note that every degradome sample has two processed and two raw data files. To have enough data, additional sequencing was performed from each sample library. And each sample raw data was processed separately (tissue_name*degradome.txt) and also combined (all_degradome*.txt).
2017-05-01 | E-GEOD-72903 | biostudies-arrayexpress
Project description:With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.
| S-EPMC3428589 | biostudies-literature
Project description:We deep sequenced a degradome library constructed from different soybean tissues. As a result, 19,830,257 represented 5,337,590 distinct signatures were obtained. 70.98% of the signatures were assigned to one soybean cDNA sequence and 24.05% matched with two cDNA sequences. 428 potential targets of small RNAs and 25 novel miRNA families were identified in soybean. A total of 211 potential miRNA targets including 150 conserved miRNA targets and 69 soybean-specific miRNA targets were identified. The signatures distribution on soybean primary miRNAs (pri-miRNAs) showed that most of the pri-miRNAs had the characteristic pattern of Dicer processing. The TAS3 small RNAs (siRNAs) biogenesis was conserved in soybean and nine Auxin Response Factors (ARFs) were identified as the TAS3 siRNA targets. The global identification of miRNAs targets would contribute to the functional research of the miRNA in soybean.
2011-11-02 | GSE33379 | GEO
Project description:We aimed to identify targets of miRNAs during wheat grain development by using degradome sequencing approach. Two degradome libraries were constructed from wheat grains. Verification of miRNA targets from two degradome libraries in developing wheat grains.
2016-07-03 | E-GEOD-65798 | biostudies-arrayexpress