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Tewari Muneesh - One of the best experts on this subject based on the ideXlab platform.

  • Phospho-RNA-Seq: a modified small RNA-Seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma
    'EMBO', 2020
    Co-Authors: Giraldez, Maria D, Spengler, Ryan M, Etheridge Alton, Goicochea, Annika J, Tuck Missy, Galas, David J, Won Choi Sung, Tewari Muneesh
    Abstract:

    Extracellular RNA s (exRNA s) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNA s in blood plasma are extensively characterized, extracellular messenger RNA (mRNA ) and long non‐coding RNA (lncRNA ) studies are limited. We report that plasma contains fragmented mRNA s and lncRNA s that are missed by standard small RNA ‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA ‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA ‐seq for sequencing synthetic RNA s with varied 5′ and 3′ ends, as well as human plasma exRNA . Analyzing phospho‐RNA ‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA /lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA ‐seq opens up new possibilities for plasma transcriptomic biomarker development.We acknowledge funding support from the NIH Extracellular RNA Communication Common Fund grants: U01 grants HL126499 to M. Tewari and HL126496 to D.J.G., and from the A. Alfred Taubman Medical Research Institute (Grand Challenge Award) to M. Tewari and S.W.C. Research reported in this publication was also supported by the National Cancer Institute of the NIH under Award Number P30CA046592 by the use of the following Cancer Center Shared Resource at the University of Michigan: DNA Sequencing. M.D.G. acknowledges support from a Precision Health Scholar Award from the University of Michigan Precision Health Center and a Juan Rodes contract (JR18/00026) funded by the Spanish Institute of Health Carlos III from the Ministry of Economy and Competitiveness (co‐funded by European Social Fund (ESF)). D.J.G. also acknowledges a special technology support award and partial funding from the Pacific Northwest Research Institute to his laboratory

  • Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma
    'EMBO', 2019
    Co-Authors: Giraldez, Maria D, Spengler, Ryan M, Etheridge Alton, Goicochea, Annika J, Tuck Missy, Choi, Sung Won, Galas, David J, Tewari Muneesh
    Abstract:

    Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non‐coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA‐seq for sequencing synthetic RNAs with varied 5′ and 3′ ends, as well as human plasma exRNA. Analyzing phospho‐RNA‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for plasma transcriptomic biomarker development.SynopsisA modified RNA‐seq method (Phospho‐RNA‐seq) revealed a new population of mRNA/lncRNA fragments in plasma, including ones that track with disease. This opens up new possibilities for disease detection via RNA profiling of plasma and other biofluids.Phospho‐RNA‐seq reveals a large population of mRNA and long non‐coding RNA fragments in human plasma, which are missed by standard small RNA‐seq protocols that depend on target RNAs having a 5′ P and 3′ OH.Accurate detection of plasma mRNA and lncRNA fragments requires a stringent bioinformatic analysis pipeline to avoid false positive alignments to mRNA and lncRNA genes.Phospho‐RNA‐seq identified ensembles of tissue‐specific transcripts in plasma of hematopoietic stem cell transplant patients, which show co‐expression patterns that vary dynamically and track with pathophysiological processes.By enabling access to an unexplored space of extracellular mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for monitoring health and disease via transcriptome fragment profiling of plasma and potentially other biofluids.A modified RNA‐seq method reveals a large population of mRNA/lncRNA fragments in plasma that are missed by standard small RNA‐seq protocols including ones that are associated with disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/1/embj2019101695_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/2/embj2019101695-sup-0002-EVFigs.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/3/embj2019101695.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/4/embj2019101695-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/5/embj2019101695.reviewer_comments.pd

Sandra Cortijo - One of the best experts on this subject based on the ideXlab platform.

  • ChIP-seq and RNA-Seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy
    Tree Genetics and Genomes, 2020
    Co-Authors: Noemie Vimont, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Fu Xiang Quah, David Guillaume Schöpfer, Sandra Cortijo
    Abstract:

    Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-Sequencing (RNA-Seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-Seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-Seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

  • chromatin immunoprecipitation sequencing and rna sequencing for complex and low abundance tree buds
    bioRxiv, 2018
    Co-Authors: Noemie Vimont, David Schoepfer, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Sandra Cortijo
    Abstract:

    Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique combined with RNA-Sequencing (RNA-Seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-Seq protocol for tree buds (Prunus avium L., Prunus persica, Malus x domestica Borkh.) that has also been successfully tested in Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. In this protocol, samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-Seq on the same starting material, differentiating it from previously published protocols. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

Noemie Vimont - One of the best experts on this subject based on the ideXlab platform.

  • ChIP-seq and RNA-Seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy
    Tree Genetics and Genomes, 2020
    Co-Authors: Noemie Vimont, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Fu Xiang Quah, David Guillaume Schöpfer, Sandra Cortijo
    Abstract:

    Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-Sequencing (RNA-Seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-Seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-Seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

  • chromatin immunoprecipitation sequencing and rna sequencing for complex and low abundance tree buds
    bioRxiv, 2018
    Co-Authors: Noemie Vimont, David Schoepfer, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Sandra Cortijo
    Abstract:

    Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique combined with RNA-Sequencing (RNA-Seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-Seq protocol for tree buds (Prunus avium L., Prunus persica, Malus x domestica Borkh.) that has also been successfully tested in Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. In this protocol, samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-Seq on the same starting material, differentiating it from previously published protocols. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

Abigail Elizur - One of the best experts on this subject based on the ideXlab platform.

  • deploying new generation sequencing for the study of flesh color depletion in atlantic salmon salmo salar
    BMC Genomics, 2021
    Co-Authors: Tuan Viet Nguyen, Gianluca Amoroso, Tomer Ventura, Abigail Elizur
    Abstract:

    The flesh pigmentation of farmed Atlantic salmon is formed by accumulation of carotenoids derived from commercial diets. In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. In this study, RNA sequencing (RNA-Seq) was performed to investigate the reduction in flesh pigmentation. Library preparation is a key step that significantly impacts the effectiveness of RNA sequencing (RNA-Seq) experiments. Besides the commonly used whole transcript RNA-Seq method, the 3’ mRNA-Seq method is being applied widely, owing to its reduced cost, enabling more repeats to be sequenced at the expense of lower resolution. Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3’ mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes.

  • Deploying new generation sequencing for the study of flesh color depletion in Atlantic Salmon (Salmo salar)
    'Springer Science and Business Media LLC', 2021
    Co-Authors: Tuan Viet Nguyen, Gianluca Amoroso, Tomer Ventura, Abigail Elizur
    Abstract:

    Abstract Background The flesh pigmentation of farmed Atlantic salmon is formed by accumulation of carotenoids derived from commercial diets. In the salmon gastrointestinal system, the hindgut is considered critical in the processes of carotenoids uptake and metabolism. In Tasmania, flesh color depletion can noticeably affect farmed Atlantic salmon at different levels of severity following extremely hot summers. In this study, RNA sequencing (RNA-Seq) was performed to investigate the reduction in flesh pigmentation. Library preparation is a key step that significantly impacts the effectiveness of RNA sequencing (RNA-Seq) experiments. Besides the commonly used whole transcript RNA-Seq method, the 3’ mRNA-Seq method is being applied widely, owing to its reduced cost, enabling more repeats to be sequenced at the expense of lower resolution. Therefore, the output of the Illumina TruSeq kit (whole transcript RNA-Seq) and the Lexogen QuantSeq kit (3’ mRNA-Seq) was analyzed to identify genes in the Atlantic salmon hindgut that are differentially expressed (DEGs) between two flesh color phenotypes. Results In both methods, DEGs between the two color phenotypes were associated with metal ion transport, oxidation-reduction processes, and immune responses. We also found DEGs related to lipid metabolism in the QuantSeq method. In the TruSeq method, a missense mutation was detected in DEGs in different flesh color traits. The number of DEGs found in the TruSeq libraries was much higher than the QuantSeq; however, the trend of DEGs in both library methods was similar and validated by qPCR. Conclusions Flesh coloration in Atlantic salmon is related to lipid metabolism in which apolipoproteins, serum albumin and fatty acid-binding protein genes are hypothesized to be linked to the absorption, transport and deposition of carotenoids. Our findings suggest that Grp could inhibit the feeding behavior of low color-banded fish, resulting in the dietary carotenoid shortage. Several SNPs in genes involving in carotenoid-binding cholesterol and oxidative stress were detected in both flesh color phenotypes. Regarding the choice of the library preparation method, the selection criteria depend on the research design and purpose. The 3’ mRNA-Seq method is ideal for targeted identification of highly expressed genes, while the whole RNA-Seq method is recommended for identification of unknown genes, enabling the identification of splice variants and trait-associated SNPs, as we have found for duox2 and duoxa1

Giraldez, Maria D - One of the best experts on this subject based on the ideXlab platform.

  • Phospho-RNA-Seq: a modified small RNA-Seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma
    'EMBO', 2020
    Co-Authors: Giraldez, Maria D, Spengler, Ryan M, Etheridge Alton, Goicochea, Annika J, Tuck Missy, Galas, David J, Won Choi Sung, Tewari Muneesh
    Abstract:

    Extracellular RNA s (exRNA s) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNA s in blood plasma are extensively characterized, extracellular messenger RNA (mRNA ) and long non‐coding RNA (lncRNA ) studies are limited. We report that plasma contains fragmented mRNA s and lncRNA s that are missed by standard small RNA ‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA ‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA ‐seq for sequencing synthetic RNA s with varied 5′ and 3′ ends, as well as human plasma exRNA . Analyzing phospho‐RNA ‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA /lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA ‐seq opens up new possibilities for plasma transcriptomic biomarker development.We acknowledge funding support from the NIH Extracellular RNA Communication Common Fund grants: U01 grants HL126499 to M. Tewari and HL126496 to D.J.G., and from the A. Alfred Taubman Medical Research Institute (Grand Challenge Award) to M. Tewari and S.W.C. Research reported in this publication was also supported by the National Cancer Institute of the NIH under Award Number P30CA046592 by the use of the following Cancer Center Shared Resource at the University of Michigan: DNA Sequencing. M.D.G. acknowledges support from a Precision Health Scholar Award from the University of Michigan Precision Health Center and a Juan Rodes contract (JR18/00026) funded by the Spanish Institute of Health Carlos III from the Ministry of Economy and Competitiveness (co‐funded by European Social Fund (ESF)). D.J.G. also acknowledges a special technology support award and partial funding from the Pacific Northwest Research Institute to his laboratory

  • Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma
    'EMBO', 2019
    Co-Authors: Giraldez, Maria D, Spengler, Ryan M, Etheridge Alton, Goicochea, Annika J, Tuck Missy, Choi, Sung Won, Galas, David J, Tewari Muneesh
    Abstract:

    Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non‐coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA‐seq for sequencing synthetic RNAs with varied 5′ and 3′ ends, as well as human plasma exRNA. Analyzing phospho‐RNA‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for plasma transcriptomic biomarker development.SynopsisA modified RNA‐seq method (Phospho‐RNA‐seq) revealed a new population of mRNA/lncRNA fragments in plasma, including ones that track with disease. This opens up new possibilities for disease detection via RNA profiling of plasma and other biofluids.Phospho‐RNA‐seq reveals a large population of mRNA and long non‐coding RNA fragments in human plasma, which are missed by standard small RNA‐seq protocols that depend on target RNAs having a 5′ P and 3′ OH.Accurate detection of plasma mRNA and lncRNA fragments requires a stringent bioinformatic analysis pipeline to avoid false positive alignments to mRNA and lncRNA genes.Phospho‐RNA‐seq identified ensembles of tissue‐specific transcripts in plasma of hematopoietic stem cell transplant patients, which show co‐expression patterns that vary dynamically and track with pathophysiological processes.By enabling access to an unexplored space of extracellular mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for monitoring health and disease via transcriptome fragment profiling of plasma and potentially other biofluids.A modified RNA‐seq method reveals a large population of mRNA/lncRNA fragments in plasma that are missed by standard small RNA‐seq protocols including ones that are associated with disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/1/embj2019101695_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/2/embj2019101695-sup-0002-EVFigs.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/3/embj2019101695.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/4/embj2019101695-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/5/embj2019101695.reviewer_comments.pd