Top PDF Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

In sequencing the nuclear RNA pool we were able to identify stable, nuclear-retained lncRNAs. These RNA species were found to be enriched in the nuclear fraction and many are present at low levels. They are likely to be missed in approaches that isolate total RNA as the cytoplasmic RNA pool is larger than the nuclear RNA pool. In comparing to existing sets of lncRNAs identified from total RNA we found only limited overlap with our set indicating that by isolating the nuclear pool of RNA we were able to identify novel nuclear retained transcripts that are masked by the cytoplasmic pool in other RNA-Seq studies. In support of this we found that for the 12 candidates we investigated further these RNAs were found almost exclusively in the nuclear fraction. One point of note is that in this approach, purely because we exclude candidates which overlap annotated genes, we overlook antisense and gene-overlapping lncRNAs. By inspection, such RNAs are still immediately obvious, the Kcnq1ot1 transcript being one example (Figure S12). Future experiments using strand-specific methodol- ogies will help further annotate this part of the nuclear transcriptome [103,104]. The nuclear-retained non-coding tran- scripts we identified are relatively stable and show lower association with RNAPII compared to other protein-coding genes expressed at similar levels (they are in the T sub-group). This suggests that they would be less easily identified using genome- wide techniques that identify nascent transcripts such as the GRO- Seq, NET-Seq and genome-wide nuclear run-on assays [9,95,105].
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De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

Total RNA from the hemocytes of 5 sick and 5 healthy octopuses selected from each group was extracted according to the Invitrogen protocol. After RNA extraction, samples were treated with Turbo DNase free (Ambion) to eliminate DNA. The RNA samples were purified using RNeasy Mini Kit (Qiagen), quantified using a NanoDrop ND1000 spectrophotometer and the RNA quality was assessed by Nano and Pico Chips Bioanalyzer (Agilent). A total of 1.5 m g of RNA from each of the 5 animals per group was pooled to construct the mRNA libraries according to the Illumina standard protocol. Thus, two mRNA libraries (one from the pool of sick octopus, and one from the pool of healthy octopus) were analyzed in a Genome Analyzer (GAII). In short, mRNA was purified using oligo (dT) probes and then fragmented into small pieces using divalent cations under a high temperature. The cleaved RNA fragments were used for first strand cDNA synthesis using random primers, modified and enriched for attachment to the Illumina flow cell. The two hemocyte libraries were generated using the mRNA sequencing sample preparation kit (Illumina). The libraries were validated by processing an Agilent DNA 1000 chip on a 2100 Bioanalyzer (Agilent) and quantified by qPCR using complementary primers of the library adapters with the KAPA SyBR FAST Universal qPCR kit (KAPA Biosystems). The cDNA libraries were sequenced on the Illumina sequencing platform (GAII equipped with a paired-end module) performing 105 cycles per read on two flow cell lanes.
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Characterization of the floral transcriptome of Moso bamboo (Phyllostachys edulis) at different flowering developmental stages by transcriptome sequencing and RNA-seq analysis.

Characterization of the floral transcriptome of Moso bamboo (Phyllostachys edulis) at different flowering developmental stages by transcriptome sequencing and RNA-seq analysis.

Previous studies of model plant species showed that the timing of floral induction is controlled by sophisticated regulatory networks that monitor changes in the environment, including autonomous pathway, photoperiod and circadian clock pathway, gibberellins pathway, ambient temperature pathway and age pathway [62,63]. The unusual and infrequent nature of bamboo flowering has attracted the curiosity of scientists and laypeople for centuries [64]. However, little research has been conducted about the molecular mechanism of bamboo flowering due to the difficulty of collecting flowering samples. Through the comparison of Moso bamboo genes found in this study with the NCBI and Uniprot databases, we identified at least 238 genes as homologs of known flowering-related genes from other plants (Table S3). These genes were compared with flowering genes from Oryza sativa, Hordeum vulgare, Brachypodium distachyon, Sorghum bicolor and Arabidopsis thaliana. However, we found that the genes employed in typical flowering promotion pathways (such as those autonomous pathway, ambient-temperature) and floral pathway integrator (FPI) genes (as [63,65]) were not highly expressed in these floral tissues in bamboo. Only late elongated hypocotyl (LHY), phyto- chrome-interacting facor 3 (PIF3), crytochrome-1 (CRY1), GI- GANTEA (GI) and CONSTANS (CO) genes were detected in Moso bamboo for the photoperiod and circadian clock pathway. We did not detect any genes homologous to components of the circadian clock and photoperiod pathway, such as early flowering 3 (ELF3), early flowering 4 (ELF4), circadian clock associated 1 (CCA1), timing of CAB1 (TOC1), pseudo response regulator (PRR5, PRR7 and PRR9) and phtyochrome (PHYA, PHYB) [66]. The CO gene plays a key role in integrating light and temporal information, and it is essential for determining the flowering time [67]. The CO protein consists of two zinc fingers, including a N-terminal B- domain mediating protein-protein interactions and a C-terminal CCT domain for nuclear localization [68]. In Arabidopsis, CO, as a transcription factor, promotes flowering by inducing the expression of flowering locus T (FT) [69]. Rice orthologues of the Arabidopsis genes CO and FT have been identified as Hd1 and Hd3a, respectively; and Hd1 controls rice flowering by regulating Hd3a (as [57]). In the present study, we found that 41 putative CO transcription factors were involved in flower development. However, most of them were down-regulated in floral tissues of Table 2. The genes related to transcription factor families.
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A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

LAM with a highly sensitive, linear RNA amplification method and the emerging RNA-Seq technology. As a model we used central cells of Arabidopsis thaliana from which only around 50 are formed within a flower, each of them individually enclosed by an ovule. Using LAM, we could obtain sufficient amounts of good quality RNA for a successful amplification and library preparation. We compared the data generated in this study with the transcriptome data from [3], which was measured using LAM and the ATH1 microarray. The results showed that the two transcriptome profiling technologies correlate well. Most of the genes found to be expressed in the microarray data were also present in the RNA-Seq data and the few microarray specific genes were likely false positives caused by probe specific cross- hybridization. However, using RNA-Seq we could detect more than double the amount of presumably expressed genes. Functionally, this difference was reflected in the enrichment of genes encoding for few specific (combinations of) protein domains, of which some may play an important role in cell fate determination (signal perception and transduction, chromatin remodeling, and regulation of transcription) or function of the specific cell type (defensin-like proteins), in the RNA-Seq data compared to the array data. In addition, we identified several intergenic regions which are likely to be transcribed. We further described a considerable fraction of reads aligning to introns and regions flanking annotated loci which may represent alternative transcript isoforms. Finally, we also performed a de novo assembly of short reads and briefly characterized the assembled transcrip- tome. Comparisons between the alignment- and the assembly- based approaches revealed that the results were remarkably similar in terms of sequence coverage pattern and Gene Ontology (GO) annotation, indicating that the workflow presented here is also suitable to study specific cell types from an organism lacking a reference sequence. Taken together, we successfully established an easy and reliable workflow that allows the transcriptional profiling of specific cell types, which are rare and difficult to access, with high sensitivity and resolution. The approach presented here will provide new insights into the transcriptional state of individual cell types not only of plants, but also other eukaryotes and, therefore, by elucidating cell fate decisions, will contribute to the under- standing of the molecular processes underlying the development of multicellular organisms.
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Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions.

Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions.

The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from cultured H. zea cells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection of H. zea cells with H. Armigera single-capsid nucleopolyhedrovirus (HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base for H. zea insect- HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of baculoviruses.
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Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the re- combinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variabili- ty. The mungbean chloroplast genome has a quadripartite structure including a pair of in- verted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chlo- roplast genome (R 2 =0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A com- bination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Bras- sica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymno- sperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at sin- gle sequence repeat regions with poly-A runs. It showed that transcriptional slippage togeth- er with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.
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Transcriptome sequencing and De Novo analysis of Youngia japonica using the illumina platform.

Transcriptome sequencing and De Novo analysis of Youngia japonica using the illumina platform.

mRNA-seq Library Construction for Illumina Sequencing The mRNA-seq library was constructed using an mRNA-Seq Sample Preparation Kit (Cat# RS-930-1001, Illumina Inc., San Diego, CA, USA) following the manufacturer’s instructions. The poly-(A) mRNA was isolated from the total RNA samples with poly-T oligo-attached magnetic beads. The mRNA was fragment- ed by an RNA fragmentation kit (Ambion, Austin, TX, USA) before cDNA synthesis to avoid priming bias. The cleaved RNA fragments were transcribed into first-strand cDNA using reverse transcriptase (Invitrogen, Carlsbad, CA, USA) (Invitrogen) and random hexamerprimers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H (Invitrogen). The double-stranded cDNA was end-repaired using T4 DNA polymerase (NEB), Klenowfragment (NEB), and T4 polynucleo- tide kinase (NEB). A base addition using Klenow 39 to 59 exo- polymerase (NEB) to prepare the DNA fragments for ligation to the adaptors. The products of ligation reaction were purified using a MinElute PCR Purification Kit (QIAGEN, Dusseldorf, Ger- many) (QIAGEN) following the manufacturer’s instructions, and eluted in 10 m L of QIAGEN EB buffer. The eluted adaptor- ligated fragments of the ligation reaction were separated by size on an agarose gel to select a size range of templates for downstream enrichment. The desired range of cDNA fragments was excised and retrieved using a Gel Extraction Kit (Axygen Biosciences, Central Avenue Union City, CA, USA). PCR was performed for the selective enrichment and amplification of the cDNA fragments using Phusion Master Mix (NEB) with two primers (i.e., PCR Primer PE 1.0 and PCR Primer PE 2.0) supplied by an mRNA- Seq Sample Preparation Kit (Illumina). The amplified products were purified using a QIAquick PCR Purification Kit (QIAGEN) following the manufacturer’s instructions, and eluted in 30 m L of QIAGEN EB buffer. Libraries were prepared from a 150 bp to 200 bp size selected fraction following adaptor ligation and agarose gel separation. After accurate quantitation (Qubit) of the samples the front steps processed, bridge PCR was performed on the surface of the flow cell to amplify DNA fragments as a single DNA molecule cluster (this process was carried out in the Cluster Station).The flow cell was then transferred into Hi-Seq for sequencing (HiSeqTM 2000 sequencing platform). Data analysis and base calling were performed using Illumina instrument software.
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RNA CoMPASS: a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets.

RNA CoMPASS: a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets.

High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism’s transcriptome. Further, the analysis of a biological specimen’s associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naı¨ve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt’s lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at http:// rnacompass.sourceforge.net/.
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Detection theory in identification of RNA-DNA sequence differences using RNA-sequencing.

Detection theory in identification of RNA-DNA sequence differences using RNA-sequencing.

Advances in sequencing technology have allowed for detailed analyses of the transcriptome at single-nucleotide resolution, facilitating the study of RNA editing or sequence differences between RNA and DNA genome-wide. In humans, two types of post-transcriptional RNA editing processes are known to occur: A-to-I deamination by ADAR and C-to-U deamination by APOBEC1. In addition to these sequence differences, researchers have reported the existence of all 12 types of RNA-DNA sequence differences (RDDs); however, the validity of these claims is debated, as many studies claim that technical artifacts account for the majority of these non-canonical sequence differences. In this study, we used a detection theory approach to evaluate the performance of RNA-Sequencing (RNA-Seq) and associated aligners in accurately identifying RNA-DNA sequence differences. By generating simulated RNA-Seq datasets containing RDDs, we assessed the effect of alignment artifacts and sequencing error on the sensitivity and false discovery rate of RDD detection. Overall, we found that even in the presence of sequencing errors, false negative and false discovery rates of RDD detection can be contained below 10% with relatively lenient thresholds. We also assessed the ability of various filters to target false positive RDDs and found them to be effective in discriminating between true and false positives. Lastly, we used the optimal thresholds we identified from our simulated analyses to identify RDDs in a human lymphoblastoid cell line. We found approximately 6,000 RDDs, the majority of which are A-to-G edits and likely to be mediated by ADAR. Moreover, we found the majority of non A-to-G RDDs to be associated with poorer alignments and conclude from these results that the evidence for widespread non-canonical RDDs in humans is weak. Overall, we found RNA-Seq to be a powerful technique for surveying RDDs genome-wide when coupled with the appropriate thresholds and filters.
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Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Mammalian Dicer was initially characterized as an exclusively cytoplasmic protein [25,26], but recent reports have challenged this localization [27]. Dicer was shown to interact with nuclear pore components and engage in nucleocytoplasmic shuttling [28]. An interaction of Dicer with ribosomal DNA repeats was demonstrated, however, a specific function could not be identified [29]. Furthermore, a large fraction of human Dicer was detected in the nucleus of HEK293 cells, where it was shown to cleave dsRNA, failure of which results in cell death due to the accumulation of dsRNA and consequent activation of the interferon response [30]. In both mouse and human cell lines, an additional nuclear role for Dicer was shown in the termination of transcription [31]. R-loops in the vicinity of a terminator induce antisense transcription and the formation of dsRNA that in turn recruits Dicer [31]. Dicer-mediated processing of this ter- minator-associated dsRNA results in the loading of Argonaute proteins with small RNAs and the subsequent recruitment of G9a and H3K9me2 at terminators, which reinforces RNA poly- merase II (Pol II) pausing and transcriptional termination [31]. In addition, Dicer was identi- fied as a regulator of alternative cleavage and polyadenylation of pre-mRNA in the nucleus of HEK293 cells [32]. Doyle et al. show that the double-stranded RNA binding domain (dsRBD)
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Pseudo-messenger RNA: phantoms of the transcriptome.

Pseudo-messenger RNA: phantoms of the transcriptome.

NMD is a phenomenon whereby mRNA molecules with nonsense (premature termination) codons undergo rapid degradation. However, the stop codons must lie at least 50–55 nt upstream of an intron. Interestingly, it has been proposed that NMD has a universal role in proofreading mRNA, protecting the cell against potentially toxic dominant negative truncated proteins. Such dominant negatives could arise for example when a ligand-binding domain is preserved but the signal transduction domain is absent [23]. It has also been shown that in the absence of NMD, the cell over- expresses transcripts arising from retroviral and retroposed elements [24]. The FANTOM dataset is expected to contain few NMD transcripts, as these are rapidly degraded and, therefore, not likely to be selected for during cloning and full-length sequencing. We assessed how many wmRNAs may be subject to NMD by checking for premature stop codons 55 nt or more upstream of a splice junction. Supposing that either the maximal ORFs or earliest ORFs are utilised, a fairly small minority of wmRNAs appear subject to NMD: 1,228 and 2,077, respectively (Figure 2). In fact, the maximal and earliest ORFs are distinct in only 2,185 cases; in these cases 1,042 earliest ORFs and 193 maximal ORFs satisfy the NMD criterion. This large discrepancy suggests that if a wmRNA is translated, the maximal ORF is more likely to be utilised.
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A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife.

A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife.

The 16S metagenomics approach was performed for each individual bank vole sample (190 in total). To obtain sequence data, two different NGS platforms were used: the Roche 454 GS-FLX pyrosequencing, or the Illumina MiSeq system (Fig 1). For 454-pyrosequencing, PCR amplification was performed on each rodent DNA sample using universal primers modified from Claesson et al. [21] (520-F: AYTGGGYDTAAAGVG; 802-R: TACCVGGGTATCTAA TCC). These amplified the V4 hypervariable region of the bacterial 16S ribosomal RNA gene (16S rRNA), generating a 207 bp product, excluding primers. Amplicon lengths were designed to be comparable with MiSeq amplicons. Primers were tagged by adding 7 bp multiplex identi- fier sequences (MIDs) and 30 bp Titanium adapters to 5’ ends as described by Galan et al. [22]. Such adapters were required for emulsion PCR (emPCR) and subsequent 454 GS-FLX pyrose- quencing using Lib-L Titanium Series reagents. We used the unique combination of 18 for- ward- and 16 reverse-primers containing distinct MIDs that permitted the amplification and individual tagging of 288 different 16S-amplicons. The tagged amplicons were then pooled, purified by AMPure XP beads (Beckman Coulter, CA, US), size selected by Pippin Prep elec- trophoresis (Sage Science, MA, USA), clonally amplified by emPCR and sequenced on a Roche 454 GS-FLX quarter picotiter plate. 454-pyrosequencing was subcontracted to Beckman Coul- ter Genomics (Danvers, MA, USA). For Illumina MiSeq sequencing, rodent DNA samples were amplified using universal primers modified from Kozich et al. [23] (16S-V4F: GTGCC AGCMGCCGCGGTAA; 16S-V4R: GGACTACHVGGGTWTCTAATCC), to amplify the bac- terial 16S rRNA V4 hypervariable region, generating 251 bp products, excluding primers. These primers were dual-indexed by adding 8 bp-indices (i5 and i7) and Nextera Illumina adaptors (P5 and P7) as described by Kozich et al. [23]. We used a unique combination of 24 i5-indeces and 36 i7-indeces, this accredit the identification and hence the ability to multiplex 864 different amplicons. The pooled amplicon library was size-selected by excision following low-melting agarose gel electrophoresis and purified using the NucleoSpin Gel clean-up kit (Macherey-Nagel, PA, USA). DNA quantification was performed by quantitative PCR using the KAPA library quantification Kit (KAPA BioSystems, MA, USA) on the final library, prior to loading on the Illumina MiSeq flow-cell using a 500 cycle reagent cartridge and 2 x 251 bp paired-end sequencing.
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De novo sequencing and characterization of the floral transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae).

De novo sequencing and characterization of the floral transcriptome of Dendrocalamus latiflorus (Poaceae: Bambusoideae).

The cDNA libraries were prepared according to the manufac- turer’s instructions (Illumina, San Diego, CA). Poly-A mRNA molecules were purified using Sera-mag Magnetic Oligo (dT) Beads (Illumina) from 20 m g total RNA from each sample and eluted with 10 mM Tris–HCl. To avoid priming bias during cDNA synthesis, the purified mRNA was first fragmented into small pieces using RNA Fragmentation Reagents (Ambion, Austin, TX, USA) before cDNA synthesis. The cleaved mRNA fragments were converted to double-stranded cDNA using random hexamer primers (Illumina) with the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA). The resulting cDNAs were purified using the QiaQuick PCR Purification Kit (Qiagen, Valencia, CA) and then subjected to end-repair and phosphory- lation using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK (NEB, Ipswich, MA, USA). Repaired cDNA fragments were 3 9 adenylated using Klenow Exo- (Illumina), producing cDNA fragments with a single ‘A’ base overhang at their 3 9 ends for subsequent adapter ligation. Illumina paired-end adapters were ligated to the ends of these 39 adenylated cDNA fragments. To select a size range of templates for downstream enrichment, the products of the ligation reaction were purified on a 2% TAE- agarose gel (Certified Low-Range Ultra Agarose, BioRad, Hercules, CA). A range of cDNA fragments (200625 bp) was excised from the gel and extracted using QIAquick Gel Extraction Kit (Qiagen). Fifteen rounds of PCR amplification were performed to enrich the purified cDNA template using primers complemen- tary to the ends of the adapters [PCR Primer PE 1.0 and PCR Primer PE 2.0 (Illumina) with Phusion DNA Polymerase. Finally, after validating on an Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 chip kit, the cDNA library products were sequenced on a paired-end flow cell using an Illumina Genome Analyzer II at Beijing Genomics Institute (BGI) in Shenzhen, China.
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Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which pre- sumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging pro- teins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting part- ners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix- domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not acti- vate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes impli- cated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regu- lation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.
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Dicer is required for haploid male germ cell differentiation in mice.

Dicer is required for haploid male germ cell differentiation in mice.

The most prominent spermatogenic defects in our Dicer1 knockout mice were found during the elongation phase of haploid differentiation. Elongation appeared to be hindered before nuclear condensation had started. This was demonstrated by the high number of uncondensed elongating spermatids as detected by phase contrast microscopy of spermatogenic cell spreads (Fig. 4A), the presence of hyperacetylated H3-positive elongating spermatids in all the stages of seminiferous epithelial cycle (Fig. 4B) and correspondingly the low number of protamine-positive nuclei (Fig. 4C) in knockout testes. Some of the germ cells were able to undergo condensation as evidenced by the presence of condensed nuclei among both testicular elongating spermatids and mature sperm (Figs. 4,5,6). However, the head morphology of these cells never appeared normal and nuclei were small and misshapen; defects in the tail development were also frequently observed. Interestingly, we demonstrate that the polarized localization of histone 1 variant H1T2 was disrupted in Dicer1 knockout mice, with H1T2 localizing on both the apical and basal sides of the nucleus. H1T2 is considered to be a molecular marker that can reveal chromatin disorganization in round and elongating spermatids [38]. H1t2 knockout mice show delayed nuclear condensation and aberrant elongation of spermatids [33]. A bipolar H1T2 mislocalization similar to the one observed in Dicer1 knockout spermatids, is also detected in testes devoid of Trf2, a transcriptional regulator involved in nuclear chromatin organiza- tion [38,39]. Therefore, our results suggest that Dicer-dependent pathways are involved in the control of chromatin architecture in haploid male germ cells.
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European sea bass (Dicentrarchus labrax) skin and scale transcriptomes

European sea bass (Dicentrarchus labrax) skin and scale transcriptomes

3 species. The sea bass genome (Tine et al. 2014) and the transcriptomes of several tissues (Louro et al. 2010; Louro et al. 2014) have been fully sequenced, mainly by Sanger sequencing of expressed sequence tag (ESTs) libraries or by 454 pyrosequencing. However, until now there are few available transcripts for the skin and scale tissues.

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Selection of the temperature of casting the bronzes to plaster moulds

Selection of the temperature of casting the bronzes to plaster moulds

Bronzes after melting were overheated to the temperature suita- bly: 1200 °C, 1180 °C, 1160 °C and 1140 °C, and then the mould was casted - probe TDAg and plaster mould with thin-walled, so- called casts test slats about dimensions: the length L=100 mm, width A=15 mm, thickness B ={4,0.8,0.5} mm (Fig. 2). Plaster moulds were executed from the mixture PRIMA-CAST about the water-plaster relation 0.4. The solidification modulus of the sections of casts, shaped in the niches of the plaster mould imitat- ing test slats were qualified from example (4):
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Transcriptome response to elevated atmospheric CO2 concentration in the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)

Transcriptome response to elevated atmospheric CO2 concentration in the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)

C. formosanus, a worldwide important pest, has been studied extensively in omics, including genome, transcriptome, metabolome, DNA methylome, and 16S rRNA sequencing (Scharf, 2015). While most studies have focused on symbionts, a few have combined host and symbiont, considering the whole termite (Scharf, 2015). Those studies are mainly based on conventional Sanger sequencing; rarely has Illumina high-throughput sequencing study been reported to date. Compare to the study by Zhang et al. (2012) using Sanger sequencing, the present study newly assembled transcriptome contains massive amounts of data (11.02 GB) using Illumina sequencing, and covers different developmental stages and castes (larva, worker, pre-soldier, soldier, reproductive). The genetic information will facilitate future developmental and caste differential studies of C. formosanus, and contribute to future work in termite comparative genomics.
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Genomewide transcriptional reprogramming in the seagrass Cymodocea nodosa under experimental ocean acidification

Genomewide transcriptional reprogramming in the seagrass Cymodocea nodosa under experimental ocean acidification

Table S4. Full annotation of C. nodosa de novo generated transcriptome. For each contig, length, first HSP result (lowest e-value), as given from the BLASTx output against Swiss- Prot and UniRef90, and related description are reported. Domain composition of putative proteins, as given by Rpstblastn against the Conserved Domains Database (CDD) is also indicated. For each sequence, the longest ORF (Open Reading Frame) and a non-coding potential score are specified. Annocript also associates the best scored putative proteins to GO terms, Enzyme Commission identifiers from the ExPASy database, and UniPathways. Table S5. Full list of significantly DEGs (FC > ±2 and FDR ≤ 0.05) in C. nodosa under elevated pCO 2 (1,200 μatm) relative to control condition (400 μatm) after 15 days of
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Maize gene atlas developed by RNA sequencing and comparative evaluation of transcriptomes based on RNA sequencing and microarrays.

Maize gene atlas developed by RNA sequencing and comparative evaluation of transcriptomes based on RNA sequencing and microarrays.

Transcriptome analysis is a valuable tool for identification and characterization of genes and pathways underlying plant growth and development. We previously published a microarray-based maize gene atlas from the analysis of 60 unique spatially and temporally separated tissues from 11 maize organs [1]. To enhance the coverage and resolution of the maize gene atlas, we have analyzed 18 selected tissues representing five organs using RNA sequencing (RNA-Seq). For a direct comparison of the two methodologies, the same RNA samples originally used for our microarray-based atlas were evaluated using RNA-Seq. Both technologies produced similar transcriptome profiles as evident from high Pearson’s correlation statistics ranging from 0.70 to 0.83, and from nearly identical clustering of the tissues. RNA-Seq provided enhanced coverage of the transcriptome, with 82.1% of the filtered maize genes detected as expressed in at least one tissue by RNA- Seq compared to only 56.5% detected by microarrays. Further, from the set of 465 maize genes that have been historically well characterized by mutant analysis, 427 show significant expression in at least one tissue by RNA-Seq compared to 390 by microarray analysis. RNA-Seq provided higher resolution for identifying tissue-specific expression as well as for distinguishing the expression profiles of closely related paralogs as compared to microarray-derived profiles. Co-expression analysis derived from the microarray and RNA-Seq data revealed that broadly similar networks result from both platforms, and that co-expression estimates are stable even when constructed from mixed data including both RNA-Seq and microarray expression data. The RNA-Seq information provides a useful complement to the microarray-based maize gene atlas and helps to further understand the dynamics of transcription during maize development.
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