RNA‐Seq Library Construction Methods for Transcriptome Analysis

Bivens, Nathan J.; Zhou, Mingyi

doi:10.1002/cppb.20019

Cited by 9 publications

(5 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Total RNA was extracted using TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Complementary DNA (cDNA) libraries were constructed following four steps including enrichment of mRNA, fragmentation of mRNA, synthesis of cDNA and the connection of an adaptor 28 . Samples were sequenced on the Illumina Nova6000 platform (Illumina, San Diego, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Temporospatial modulation of Lymantria dispar immune system against an entomopathogenic fungal infection

Bai

et al. 2020

Pest Management Science

View full text Add to dashboard Cite

BACKGROUND: Lymantria dispar is an economically impactful forest pest worldwide. The entomopathogenic fungi Beauveria bassiana shows great promise in pest management due to its high lethality in Lymantria dispar. A complete understanding of the immune interactions between the pest and the pathogenic fungus is essential to actualizing biological pest management. RESULTS: Following the infection of Lymantria dispar by Beauveria bassiana spores, we performed a time-course analysis of transcriptome in Lymantria dispar fat bodies and hemocytes to explore host immune response. A total of 244 immunity-related genes including pattern recognition receptors, extracellular signal modulators, immune pathways (Toll, IMD, JNK and JAK/ STAT), and response effectors were identified. We observed contrasting tissue and time-specific differences in the expression of immune genes. At the early stage of infection, several recognition receptors and effector genes were activated, while the signal modulation and effector genes were suppressed at later stages. Further enzyme activity-based assays coupled with gene expression analysis of prophenoloxidase revealed a significant upregulation of phenoloxidase activity at 48-and 72-h postinfection. Moreover, fungal infection led to dysbiosis in gut microbiota that seems to be partially attributed to reduced gut hydrogen peroxide (H 2 O 2) amount, which indicates a significant impact of fungal infection on host gut microbes. CONCLUSION: Our study provides a comprehensive sequence resource and crucial new insights about an economically important forest pest. Specifically, we elucidate the complicated multipartite interaction between host and fungal pathogen and contribute to a better understanding of Lymantria dispar anti-fungal immunity, resulting in better tools for biological pest control.

show abstract

Section: Methodsmentioning

confidence: 99%

Temporospatial modulation of Lymantria dispar immune system against an entomopathogenic fungal infection

Bai

et al. 2020

Pest Management Science

View full text Add to dashboard Cite

show abstract

“…As a revolutionary tool, RNA-Seq technology gives the opportunity to produce large numbers of sequence data in non-model organisms [ 33 , 34 ]. The transcriptional and proteomics landscape in the host upon virus infection facilitates the understanding of host immune responses and defense mechanisms based on the pathogenic microorganism infection at the whole mRNA and protein level and provides new approaches to control the viral infections.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

Fei

Xie

Liu

et al. 2021

Viruses

View full text Add to dashboard Cite

Caprine herpesvirus 1 (CpHV-1) is a member of the alpha subfamily of herpesviruses, which is responsible for genital lesions and latent infections in goat populations worldwide. In this study, for the first time, the transcriptome and proteomics of CpHV-1 infected Madin Darby bovine kidney (MDBK) cells were explored using RNA-Sequencing (RNA-Seq) and isobaric tags for relative and absolute quantitation-liquid chromatography tandem mass spectrometry (iTRAQ-LC-MS/MS) technology, respectively. RNA-Seq analysis revealed 81 up-regulated and 19 down-regulated differentially expressed genes (DEGs) between infected and mock-infected MDBK cells. Bioinformatics analysis revealed that most of these DEGs were mainly involved in the innate immune response, especially the interferon stimulated genes (ISGs). Gene Ontology (GO) enrichment analysis results indicated that the identified DEGs were significantly mainly enriched for response to virus, defense response to virus, response to biotic stimulus and regulation of innate immune response. Viral carcinogenesis, the RIG-I-like receptor signaling pathway, the cytosolic DNA-sensing pathway and pathways associated with several viral infections were found to be significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Eleven selected DEGs (Mx1, RSAD2, IFIT1, IFIT2, IFIT5, IFIH1, IFITM3, IRF7, IRF9, OAS1X and OAS1Y) associated with immune responses were selected, and they exhibited a concordant direction both in RNA-Seq and quantitative real-time RT-PCR analysis. Proteomic analysis also showed significant up-regulation of innate immunity-related proteins. GO analysis showed that the differentially expressed proteins were mostly enriched in defense response and response to virus, and the pathways associated with viral infection were enriched under KEGG analysis. Protein-protein interaction network analysis indicated most of the DEGs related to innate immune responses, as DDX58(RIG-I), IFIH1(MDA5), IRF7, Mx1, RSAD2, OAS1 and IFIT1, were located in the core of the network and highly connected with other DGEs. Our findings support the notion that CpHV-1 infection induced the transcription and protein expression alterations of a series of genes related to host innate immune response, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of CpHV-1.

show abstract

“…(∼0.6 - 1.1 ug total). ds cDNA samples were sent to the University of Missouri Genomics Technology Core, where library preparation was performed (average insert size of 500 bp) using the Illumina TruSeq DNA PCR-Free Library Prep Kit according to the protocol described in (100). 250 bp paired-end sequencing was performed at the Tufts University Genomics Core on an Illumina HiSeq 2500 machine in Rapid Run mode.…”

Section: Methodsmentioning

confidence: 99%

Predicting yield traits of individual field-grownBrassica napusplants from rosette-stage leaf gene expression

Meyer

Cruz

Swaef

et al. 2022

Preprint

View full text Add to dashboard Cite

BackgroundIn the plant sciences, results of laboratory studies often do not translate well to the field because lab growth conditions are very different from field conditions. To help close this lab-field gap, we developed a new strategy for studying the wiring of plant traits directly in the field, based on molecular profiling and phenotyping of individual plants of the same genetic background grown in the same field. This single-plant omics strategy leverages uncontrolled micro-environmental variation across the field and stochastic variation among the individual plants as information sources, rather than controlled perturbations. Here, we use single-plant omics on winter-typeBrassica napus(rapeseed) plants to investigate to what extent rosette-stage gene expression profiles can be linked to the early and late phenotypes of individual field-grown plants.ResultsWe find that rosette leaf gene expression in autumn has substantial predictive power for both autumnal leaf phenotypes and final yield in spring. Many of the top predictor genes are linked to developmental processes known to occur in autumn in winter-typeB. napusaccessions, such as the juvenile-to-adult and vegetative-to-reproductive phase transitions, indicating that the yield potential of winter-typeB. napusis influenced by autumnal development.ConclusionsOur results show that profiling individual plants under uncontrolled field conditions is a valid strategy for identifying genes and processes influencing crop yield in the field.

show abstract

RNA‐Seq Library Construction Methods for Transcriptome Analysis

Cited by 9 publications

References 33 publications

Temporospatial modulation of Lymantria dispar immune system against an entomopathogenic fungal infection

Temporospatial modulation of Lymantria dispar immune system against an entomopathogenic fungal infection

Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

Predicting yield traits of individual field-grownBrassica napusplants from rosette-stage leaf gene expression

Contact Info

Product

Resources

About