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Sophie Dumont - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell
2016Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Austra-lia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell bio-logical processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat kan-garoo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcrip-tome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcrip-tome will make Rat Kangaroo cells a more tractable system for linking molecular-scale func-tion and cellular-scale dynamics
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draft de novo transcriptome of the Rat Kangaroo potorous tridactylus as a tool for cell biology
PLOS ONE, 2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat Kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make Rat Kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.
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Rat Kangaroo transcriptome-wide analysis.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Flow chart indicating the numbers of and relationships among coding and non-coding transcripts and Unigenes. Labels S1-S4 indicate from which datasets Supporting Information S1–S4 files arise. (B) Distributions of RSEM-inferred TPM values for: all protein coding (blue) or non-coding (red) transcripts (solid) or Unigenes (dashed) in the Rat Kangaroo transcriptome. These colors and line styles are consistently used in (A). (C) Distributions of protein coding Unigene TPMs (as in (B)) and the subset of protein coding Unigenes annotated by GO biological process terms (black solid). (D) The 30 GO biological process terms occurring most frequently in the annotation of the 20,079 Unigenes are listed with number of Unigenes annotated by each term (left) and distribution of RSEM gene-level abundance estimates for the annotated Unigenes (right; boxes indicate interquartile range (IQR), red bars indicate medians, whiskers indicate most extreme data points within 1.5*IQR of the boxes, and crosses indicate outliers).
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Proof-of-concept experimental validation of Rat Kangaroo transcriptome: single gene analysis (PRC1).
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Sequence of the 19 bp siRNA (siPRC1-A) designed against the Rat Kangaroo PRC1 sequence from the transcriptome. This siRNA was used in subsequent experiments to validate the siRNA design protocol and confirm successful knockdown. (B) Immunoblot demonstRating successful depletion of PRC1 protein after RNAi. Here and forward, “mock control” represents an Oligofectamine-only control. The tubulin band serves as a loading control. (C) Percentage of binucleated cells in a population of control and siRNA-treated cells 48 h after treatment. In the control population, 2/278 cells were binucleated (0.7%). In the siRNA-treated population, 53/256 cells were binucleated (20.7%). (D) Immunofluorescence image showing phenotype during cytokinesis in control and siRNA-treated cells. PRC1 localizes at the spindle midzone in control cells during cytokinesis, and is absent from the midzone during cytokinesis in siRNA-treated cells. (E) Immunofluorescence image showing the interphase localization of PRC1 in control and siRNA-treated cells. Control cells show nuclear localization of PRC1 and are mononucleated, while siRNA-treated cells show no PRC1 localization and are often binucleated. (F) Time strip showing cytokinesis failure for siRNA-treated cells. Phase contrast (top) and GFP-tubulin (bottom) images are shown. Cytokinesis appears to be successful at 1:04:01, but at 1:30:01 it becomes clear that the daughter cells never completely sepaRated and they have come back together to form one binucleated cell. In total, 16 cells were imaged through cytokinesis in this manner, and four appeared to undergo this type of failed cytokinesis, a Ratio similar to that revealed by counting of binucleated cells by immunofluorescence (C). Time in h:min:sec.
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Single gene analysis (PRC1): Within- and cross-species transcript isoform comparisons.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:Alignment of all PRC1 mRNA isoforms from Rat Kangaroo, gray short-tailed opossum, and human relative to the human genomic PCR1 DNA sequence, providing a basis for within- and cross-species putative splicing isoform comparisons. The numbered boxes correspond to exons, while the horizontal lines in between exons correspond to introns. Exons that change in length or are absent within or between species are colored for emphasis, with darker colors marking longer exon isoforms within a given exon (length differences may be only a few amino acids). The target location of the chosen siRNA PRC1-A for the proof-of-concept experiment presented below is marked by “+”, and the target locations of other tested siRNAs are marked by “*”. While the overall sequence similarity is much higher between Rat Kangaroo and opossum than Rat Kangaroo and human, the inferred structures are highly concordant among all three species.
Dylan B Udy - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell
2016Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Austra-lia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell bio-logical processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat kan-garoo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcrip-tome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcrip-tome will make Rat Kangaroo cells a more tractable system for linking molecular-scale func-tion and cellular-scale dynamics
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draft de novo transcriptome of the Rat Kangaroo potorous tridactylus as a tool for cell biology
PLOS ONE, 2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat Kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make Rat Kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.
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Rat Kangaroo transcriptome-wide analysis.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Flow chart indicating the numbers of and relationships among coding and non-coding transcripts and Unigenes. Labels S1-S4 indicate from which datasets Supporting Information S1–S4 files arise. (B) Distributions of RSEM-inferred TPM values for: all protein coding (blue) or non-coding (red) transcripts (solid) or Unigenes (dashed) in the Rat Kangaroo transcriptome. These colors and line styles are consistently used in (A). (C) Distributions of protein coding Unigene TPMs (as in (B)) and the subset of protein coding Unigenes annotated by GO biological process terms (black solid). (D) The 30 GO biological process terms occurring most frequently in the annotation of the 20,079 Unigenes are listed with number of Unigenes annotated by each term (left) and distribution of RSEM gene-level abundance estimates for the annotated Unigenes (right; boxes indicate interquartile range (IQR), red bars indicate medians, whiskers indicate most extreme data points within 1.5*IQR of the boxes, and crosses indicate outliers).
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Proof-of-concept experimental validation of Rat Kangaroo transcriptome: single gene analysis (PRC1).
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Sequence of the 19 bp siRNA (siPRC1-A) designed against the Rat Kangaroo PRC1 sequence from the transcriptome. This siRNA was used in subsequent experiments to validate the siRNA design protocol and confirm successful knockdown. (B) Immunoblot demonstRating successful depletion of PRC1 protein after RNAi. Here and forward, “mock control” represents an Oligofectamine-only control. The tubulin band serves as a loading control. (C) Percentage of binucleated cells in a population of control and siRNA-treated cells 48 h after treatment. In the control population, 2/278 cells were binucleated (0.7%). In the siRNA-treated population, 53/256 cells were binucleated (20.7%). (D) Immunofluorescence image showing phenotype during cytokinesis in control and siRNA-treated cells. PRC1 localizes at the spindle midzone in control cells during cytokinesis, and is absent from the midzone during cytokinesis in siRNA-treated cells. (E) Immunofluorescence image showing the interphase localization of PRC1 in control and siRNA-treated cells. Control cells show nuclear localization of PRC1 and are mononucleated, while siRNA-treated cells show no PRC1 localization and are often binucleated. (F) Time strip showing cytokinesis failure for siRNA-treated cells. Phase contrast (top) and GFP-tubulin (bottom) images are shown. Cytokinesis appears to be successful at 1:04:01, but at 1:30:01 it becomes clear that the daughter cells never completely sepaRated and they have come back together to form one binucleated cell. In total, 16 cells were imaged through cytokinesis in this manner, and four appeared to undergo this type of failed cytokinesis, a Ratio similar to that revealed by counting of binucleated cells by immunofluorescence (C). Time in h:min:sec.
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Single gene analysis (PRC1): Within- and cross-species transcript isoform comparisons.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:Alignment of all PRC1 mRNA isoforms from Rat Kangaroo, gray short-tailed opossum, and human relative to the human genomic PCR1 DNA sequence, providing a basis for within- and cross-species putative splicing isoform comparisons. The numbered boxes correspond to exons, while the horizontal lines in between exons correspond to introns. Exons that change in length or are absent within or between species are colored for emphasis, with darker colors marking longer exon isoforms within a given exon (length differences may be only a few amino acids). The target location of the chosen siRNA PRC1-A for the proof-of-concept experiment presented below is marked by “+”, and the target locations of other tested siRNAs are marked by “*”. While the overall sequence similarity is much higher between Rat Kangaroo and opossum than Rat Kangaroo and human, the inferred structures are highly concordant among all three species.
Mark S Springer - One of the best experts on this subject based on the ideXlab platform.
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A phylogeny and timescale for the living genera of Kangaroos and kin (Macropodiformes : Marsupialia) based on nuclear DNA sequences
Australian Journal of Zoology, 2008Co-Authors: Robert W Meredith, Michael Westerman, Mark S SpringerAbstract:Kangaroos and kin (Macropodiformes) are the most conspicuous elements of the Australasian marsupial fauna. The approximately 70 living species can be divided into three families: (1) Hypsiprymnodontidae (the musky Rat Kangaroo); (2) Potoroidae (potoroos and bettongs); and (3) Macropodidae (larger Kangaroos, wallabies, banded hare wallaby and pademelons). Here we examine macropodiform relationships using protein-coding portions of the ApoB, BRCA1, IRBP, Rag1 and vWF genes via maximum parsimony, maximum likelihood and Bayesian methods. We estimate times of divergence using two different relaxed molecular clock methods to present a timescale for macropodiform evolution and reconstruct ancestral states for grades of dental organisation. We find robust support for a basal split between Hypsiprymnodontidae and the other macropodiforms, potoroid monophyly and macropodid monophyly, with Lagostrophus as the sister-taxon to all other macropodids. Our divergence estimates suggest that Kangaroos diverged from Phalangeroidea in the early Eocene, that crown-group Macropodiformes originated in the late Eocene or early Oligocene and that the potoroid–macropodid split occurred in the late Oligocene or early Miocene followed by rapid cladogenesis within these families 5 to 15 million years ago. These divergence estimates coincide with major geological and ecological changes in Australia. Ancestral state reconstructions for grades of dental organisation suggest that the grazer grade evolved independently on two different occasions within Macropodidae.
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The phylogenetic position of the musky Rat-Kangaroo and the evolution of bipedal hopping in Kangaroos (Macropodidae: Diprotodontia).
Systematic Biology, 1998Co-Authors: Angela Burk, Michael Westerman, Mark S SpringerAbstract:Kangaroos and their relatives (family Macropodidae) are divided into the subfamilies Macropodinae (Kangaroos, wallabies, pademelons) and Potoroinae (Rat-Kangaroos, potoroos, bet- tongs). The musky Rat-Kangaroo, Hypsiprymnodon moschatus , is traditionally allied with other potoroines, based primarily on the basis of osteological characters and aspects of the female reproductive system. Unlike other macropodids, however, which are capable of bipedal hopping, Hypsiprymnodon is a quadrupedal bounder and lacks several derived features of the pes and tarsus that are presumably adaptations for bipedal hopping. Other derived features, such as a complex stomach, loss of P 2 with the eruption of P 3, and reduction of litter size to one, are also lacking in Hypsiprymnodon but occur in all other macropodids. Thus, available evidence suggests that Hypsiprymnodon either is part of a monophyletic Potoroinae or is a sister taxon to other living macropodids. To test these hypotheses, we sequenced 1,170 bp base pairs of the mitochondrial genome for16 macropodids. Maximum parsimony, minimum evolution, maximum likelihood, and quartet puzzling all support the hypothesis that macropodines and potoroines are united to the exclusion of Hypsiprymnodon . This hypothesis implies that characters such as bipedal hopping evolved only once in macropodid evolution. Aside from Hypsiprymnodon , the remaining macro- podids sepaRate into the traditional Macropodinae and Potoroinae. Macropodines further sepaRate into two clades: one containing the New Guinean forest wallabies Dorcopsis and Dorcopsulus , and one consisting of the genera M acropus, Setonix, Thylogale, Onychogalea , W allabia, Dendrolagus , Peradorcas, and Lagorchestes . Among potoroines, there is modeRate support for the association of Bettongia and Aepyprymnus to the exclusion of Potorous. Divergence times were estimated by using 12S ribosomal RNA transversions. At the base of the macropodid radiation, Hypsiprymnodon divergedfrom othermacropodids approximately 45 million years ago. This estimate is comparable to divergence estimates among families of Australasian possums based on single-copy DNA hybridization and 12S rRNA transversions. Macropodines and potoroines, in turn, diverged approximately 30 million years ago. Among macropodines, Dorcopsis and Dorcopsulus sepaRated from other taxa approximately 10 million years ago. (bipedal hopping; Hypsiprymnodon ; Kangaroo; Macropodidae; ribosomal RNA)
Mark Voorhies - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell
2016Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Austra-lia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell bio-logical processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat kan-garoo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcrip-tome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcrip-tome will make Rat Kangaroo cells a more tractable system for linking molecular-scale func-tion and cellular-scale dynamics
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draft de novo transcriptome of the Rat Kangaroo potorous tridactylus as a tool for cell biology
PLOS ONE, 2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat Kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make Rat Kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.
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Rat Kangaroo transcriptome-wide analysis.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Flow chart indicating the numbers of and relationships among coding and non-coding transcripts and Unigenes. Labels S1-S4 indicate from which datasets Supporting Information S1–S4 files arise. (B) Distributions of RSEM-inferred TPM values for: all protein coding (blue) or non-coding (red) transcripts (solid) or Unigenes (dashed) in the Rat Kangaroo transcriptome. These colors and line styles are consistently used in (A). (C) Distributions of protein coding Unigene TPMs (as in (B)) and the subset of protein coding Unigenes annotated by GO biological process terms (black solid). (D) The 30 GO biological process terms occurring most frequently in the annotation of the 20,079 Unigenes are listed with number of Unigenes annotated by each term (left) and distribution of RSEM gene-level abundance estimates for the annotated Unigenes (right; boxes indicate interquartile range (IQR), red bars indicate medians, whiskers indicate most extreme data points within 1.5*IQR of the boxes, and crosses indicate outliers).
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Proof-of-concept experimental validation of Rat Kangaroo transcriptome: single gene analysis (PRC1).
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Sequence of the 19 bp siRNA (siPRC1-A) designed against the Rat Kangaroo PRC1 sequence from the transcriptome. This siRNA was used in subsequent experiments to validate the siRNA design protocol and confirm successful knockdown. (B) Immunoblot demonstRating successful depletion of PRC1 protein after RNAi. Here and forward, “mock control” represents an Oligofectamine-only control. The tubulin band serves as a loading control. (C) Percentage of binucleated cells in a population of control and siRNA-treated cells 48 h after treatment. In the control population, 2/278 cells were binucleated (0.7%). In the siRNA-treated population, 53/256 cells were binucleated (20.7%). (D) Immunofluorescence image showing phenotype during cytokinesis in control and siRNA-treated cells. PRC1 localizes at the spindle midzone in control cells during cytokinesis, and is absent from the midzone during cytokinesis in siRNA-treated cells. (E) Immunofluorescence image showing the interphase localization of PRC1 in control and siRNA-treated cells. Control cells show nuclear localization of PRC1 and are mononucleated, while siRNA-treated cells show no PRC1 localization and are often binucleated. (F) Time strip showing cytokinesis failure for siRNA-treated cells. Phase contrast (top) and GFP-tubulin (bottom) images are shown. Cytokinesis appears to be successful at 1:04:01, but at 1:30:01 it becomes clear that the daughter cells never completely sepaRated and they have come back together to form one binucleated cell. In total, 16 cells were imaged through cytokinesis in this manner, and four appeared to undergo this type of failed cytokinesis, a Ratio similar to that revealed by counting of binucleated cells by immunofluorescence (C). Time in h:min:sec.
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Single gene analysis (PRC1): Within- and cross-species transcript isoform comparisons.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:Alignment of all PRC1 mRNA isoforms from Rat Kangaroo, gray short-tailed opossum, and human relative to the human genomic PCR1 DNA sequence, providing a basis for within- and cross-species putative splicing isoform comparisons. The numbered boxes correspond to exons, while the horizontal lines in between exons correspond to introns. Exons that change in length or are absent within or between species are colored for emphasis, with darker colors marking longer exon isoforms within a given exon (length differences may be only a few amino acids). The target location of the chosen siRNA PRC1-A for the proof-of-concept experiment presented below is marked by “+”, and the target locations of other tested siRNAs are marked by “*”. While the overall sequence similarity is much higher between Rat Kangaroo and opossum than Rat Kangaroo and human, the inferred structures are highly concordant among all three species.
Todd M Lowe - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell
2016Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Austra-lia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell bio-logical processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat kan-garoo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcrip-tome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcrip-tome will make Rat Kangaroo cells a more tractable system for linking molecular-scale func-tion and cellular-scale dynamics
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draft de novo transcriptome of the Rat Kangaroo potorous tridactylus as a tool for cell biology
PLOS ONE, 2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:The Rat Kangaroo (long-nosed potoroo, Potorous tridactylus) is a marsupial native to Australia. Cultured Rat Kangaroo kidney epithelial cells (PtK) are commonly used to study cell biological processes. These mammalian cells are large, adherent, and flat, and contain large and few chromosomes—and are thus ideal for imaging intra-cellular dynamics such as those of mitosis. Despite this, neither the Rat Kangaroo genome nor transcriptome have been sequenced, creating a challenge for probing the molecular basis of these cellular dynamics. Here, we present the sequencing, assembly and annotation of the draft Rat Kangaroo de novo transcriptome. We sequenced 679 million reads that mapped to 347,323 Trinity transcripts and 20,079 Unigenes. We present statistics emerging from transcriptome-wide analyses, and analyses suggesting that the transcriptome covers full-length sequences of most genes, many with multiple isoforms. We also validate our findings with a proof-of-concept gene knockdown experiment. We expect that this high quality transcriptome will make Rat Kangaroo cells a more tractable system for linking molecular-scale function and cellular-scale dynamics.
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Rat Kangaroo transcriptome-wide analysis.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Flow chart indicating the numbers of and relationships among coding and non-coding transcripts and Unigenes. Labels S1-S4 indicate from which datasets Supporting Information S1–S4 files arise. (B) Distributions of RSEM-inferred TPM values for: all protein coding (blue) or non-coding (red) transcripts (solid) or Unigenes (dashed) in the Rat Kangaroo transcriptome. These colors and line styles are consistently used in (A). (C) Distributions of protein coding Unigene TPMs (as in (B)) and the subset of protein coding Unigenes annotated by GO biological process terms (black solid). (D) The 30 GO biological process terms occurring most frequently in the annotation of the 20,079 Unigenes are listed with number of Unigenes annotated by each term (left) and distribution of RSEM gene-level abundance estimates for the annotated Unigenes (right; boxes indicate interquartile range (IQR), red bars indicate medians, whiskers indicate most extreme data points within 1.5*IQR of the boxes, and crosses indicate outliers).
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Proof-of-concept experimental validation of Rat Kangaroo transcriptome: single gene analysis (PRC1).
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:(A) Sequence of the 19 bp siRNA (siPRC1-A) designed against the Rat Kangaroo PRC1 sequence from the transcriptome. This siRNA was used in subsequent experiments to validate the siRNA design protocol and confirm successful knockdown. (B) Immunoblot demonstRating successful depletion of PRC1 protein after RNAi. Here and forward, “mock control” represents an Oligofectamine-only control. The tubulin band serves as a loading control. (C) Percentage of binucleated cells in a population of control and siRNA-treated cells 48 h after treatment. In the control population, 2/278 cells were binucleated (0.7%). In the siRNA-treated population, 53/256 cells were binucleated (20.7%). (D) Immunofluorescence image showing phenotype during cytokinesis in control and siRNA-treated cells. PRC1 localizes at the spindle midzone in control cells during cytokinesis, and is absent from the midzone during cytokinesis in siRNA-treated cells. (E) Immunofluorescence image showing the interphase localization of PRC1 in control and siRNA-treated cells. Control cells show nuclear localization of PRC1 and are mononucleated, while siRNA-treated cells show no PRC1 localization and are often binucleated. (F) Time strip showing cytokinesis failure for siRNA-treated cells. Phase contrast (top) and GFP-tubulin (bottom) images are shown. Cytokinesis appears to be successful at 1:04:01, but at 1:30:01 it becomes clear that the daughter cells never completely sepaRated and they have come back together to form one binucleated cell. In total, 16 cells were imaged through cytokinesis in this manner, and four appeared to undergo this type of failed cytokinesis, a Ratio similar to that revealed by counting of binucleated cells by immunofluorescence (C). Time in h:min:sec.
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Single gene analysis (PRC1): Within- and cross-species transcript isoform comparisons.
2015Co-Authors: Dylan B Udy, Mark Voorhies, Patricia P Chan, Todd M Lowe, Sophie DumontAbstract:Alignment of all PRC1 mRNA isoforms from Rat Kangaroo, gray short-tailed opossum, and human relative to the human genomic PCR1 DNA sequence, providing a basis for within- and cross-species putative splicing isoform comparisons. The numbered boxes correspond to exons, while the horizontal lines in between exons correspond to introns. Exons that change in length or are absent within or between species are colored for emphasis, with darker colors marking longer exon isoforms within a given exon (length differences may be only a few amino acids). The target location of the chosen siRNA PRC1-A for the proof-of-concept experiment presented below is marked by “+”, and the target locations of other tested siRNAs are marked by “*”. While the overall sequence similarity is much higher between Rat Kangaroo and opossum than Rat Kangaroo and human, the inferred structures are highly concordant among all three species.
