An improved protocol for small RNA library construction by using High-Definition adapters [cell line]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Irina Mohorianu
PROVIDER: E-GEOD-59331 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of medicago truncatula leaves were constructed by using an improved protocol where high-definition (HD) adapters were used.
2015-01-31 | E-GEOD-59330 | biostudies-arrayexpress
Project description:The small RNA libraries from Moso bamboo (Phyllostachy heterocycla) roots and leaves were constructed by using high definition adapters . The small RNA profiles were analyzed. A collection of micro RNAs with similarity to the micro RNA entries in mirbase were discovered. The putative genomic loci of the micro RNAs were identified. Analysis of small RNA profiles from the root and leaf tissues of young Moso Bamboo seedlings
2014-08-01 | E-GEOD-55305 | biostudies-arrayexpress
Project description:Purpose: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing. Results: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAIRY MERISTEM (HAM) respectively, were experimentally validated using 5M-bM-^@M-2RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions. Conclusions: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber developmental process. Total seven (Leaf, Root, Stem, Potato Tuber stage 0(PT0),Potato Tuber stage 1(PT1),Potato Tuber stage 2(PT2),Potato Tuber stage 3(PT3) ) small RNA libraries were consctructed and sequenced by deep sequencing using Illumina GAIIx.
2014-01-09 | E-GEOD-52599 | biostudies-arrayexpress
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of chondrosarcoma cell line. The adapter dimer was significantly reduced. The small RNA sequences were also analysed.
2015-01-31 | GSE59331 | GEO
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed.
2015-01-31 | GSE59330 | GEO
Project description:Purpose: To identify abiotic stress responsive and tissue specific miRNAs at genome wide level in wheat (Triticum aestivum) Results: Small RNA libraries were constructed from four tissues (root, shoot, mature leaf and spikelets) and three stress treatments of wheat seedlings (control, high temperature, salinity and water-deficit). A total of 59.5 million reads were obtained by high throughput sequencing of eight wheat libraries, of which 32.5 million reads were found to be unique. Using UEA sRNA workbench we identified 47 conserved miRNAs belonging to 20 families, 1030 candidate novel and 51 true novel miRNAs. Several of these miRNAs displayed tissue specific expression whereas few were found to be responsive to abiotic stress treatments. Target genes were predicted for miRNAs identified in this study and their grouping into functional categories revealed that the putative targets were involved in diverse biological processes. RLM-RACE of predicted targets of three conserved miRNAs (miR156, miR160 and miR164) confirmed their mRNA cleavage, thus indicating their regulation at post-transcriptional level by corresponding miRNAs. Expression profiling of confirmed target genes of these miRNAs was also performed. Conclusions: This is the first comprehensive study on profiling of miRNAs in a variety of tissues and in response to several abiotic stresses in wheat. Our findings provide valuable resource for better understanding on the role of miRNAs in stress tolerance as well as plant development. Additionally, this information could be utilized for designing wheat plants for enhanced abiotic stress tolerance and higher productivity. Total eight (three stress, one control and four tissue specific small RNA libraries were pepared and sequenced independently [wheat control (WC), wheat high temperature stressed (WHTS), wheat salinity stressed (WSS) and wheat drought stressed (WDS), wheat shoot(WSH), wheat leaf (WLF), wheat flower(WFL), wheat root(WRT)] on Illumina GAIIx
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Project description:MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation. miRNAs have been shown to control many genes involved in various biological and metabolic processes. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants. In this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues, Based on sequence similarity and hairpin structure prediction, we found that 178,102 reads representing 89 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates, whose precursors were all potentially generated from citrus ESTs. And of them five miRNA* sequences were also sequenced. These sequences had not been described in other plant species and accumulation of these 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes included one encoding IRX12 copper ion binding/ oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata. Size fractionated small RNAs (16-30 bp) from total RNA extracts was ligated to 5' and 3' adapters, and reverse transcribed. After PCR amplification the sample was subjected to Solexa sequencing. The resultant 35nt sequence data were filtered according to base quality value. The remained sequences were used to trim 5' and 3' adaptors. The clean tags were used for further analysis.
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2013-08-01 | E-GEOD-47886 | biostudies-arrayexpress