Direct-TRI: High-throughput RNA-extracting Method for all Stages of Zebrafish Development

Ujibe, Kota; Nishimura, Koshi; Kashima, Makoto; Hirata, Hiromi

doi:10.21769/bioprotoc.4136

Cited by 9 publications

(3 citation statements)

References 15 publications

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“…Total RNA was extracted from each individual planarian using the “Direct-TRI” method [19]. Briefly, a planarian was lysed using TRI Reagent-LS (Molecular Research Center, Cincinnati, OH, USA).…”

Section: Methodsmentioning

confidence: 99%

Tensor Decomposition-based Unsupervised Feature Extraction Succeeded in Identification of Differentially Expressed Transcripts from Redundantde novoTranscriptome ofPlanarian

Kashima

Kumagai

Hirata

et al. 2021

Preprint

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RNA-Seq data analysis of non-model organisms is often difficult because of the lack of a well-annotated genome. In model organisms, after short reads are mapped to the genome, it is possible to focus on the analysis of regions well-annotated regions. However, in non-model organisms, contigs can be generated by de novo assembling. This can result in a large number of transcripts, making it difficult to easily remove redundancy. A large number of transcripts can also lead to difficulty in the recognition of differentially expressed transcripts (DETs) between more than two experimental conditions, because P-values must be corrected by considering multiple comparison corrections whose effect is enhanced as the number of transcripts increases. Heavily corrected P-values often fail to take sufficiently small P-values as significant. In this study, we applied a recently proposed tensor decomposition (TD)-based unsupervised feature extraction (FE) to the RNA-seq data obtained for a non-model organism, Planarian; we successfully obtained a limited number of transcripts whose expression was altered between normal and defective samples as well as during time development. TD-based unsupervised FE is expected to be an effective tool that can identify a limited number of DETs, even when a poorly annotated genome is available.

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Section: Methodsmentioning

confidence: 99%

Tensor Decomposition-based Unsupervised Feature Extraction Succeeded in Identification of Differentially Expressed Transcripts from Redundantde novoTranscriptome ofPlanarian

Kashima

Kumagai

Hirata

et al. 2021

Preprint

Self Cite

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“…Using morpholino interference technology [10], we successfully obtained the asb5a −/− :asb5b-ATGmo double-knockout zebrafish strain. RNA sequencing [11,12] was used to analyze the transcriptome differences between the asb5a −/− :asb5b-ATGmo double-knockout group and the wild-type (WT) group, as well as the asb5a −/− single-knockout group, respectively. We found that the double-knockout group was significantly enriched in functional items related to cardiac muscle contraction.…”

Section: Introductionmentioning

confidence: 99%

asb5a/asb5b Double Knockout Affects Zebrafish Cardiac Contractile Function

Cai,

Wang,

Luo

et al. 2023

IJMS

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Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two asb5 gene isoforms, asb5a and asb5b. Currently, the effects of asb5 gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated asb5a-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar asb5a and asb5b expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target asb5b in wild-type and asb5a-knockout zebrafish. Knocking down asb5b in the wild-type had no phenotypic impact, but simultaneous asb5b knockdown in asb5a-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in asb5a and asb5b, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into asb5’s mechanism.

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“…Recent technical advances related to bulk RNA-Seq have overcome these limitations. Advances in sequencing platforms ( Muir et al, 2016 ), RNA extraction method ( Yoshino et al, 2020 ; Ujibe et al, 2021 ), and bulk 3′ RNA-Seq library preparation methods ( Alpern et al, 2019 ; Kamitani et al, 2019 ; Li et al, 2020 ) have enabled a cost-effective time-course individual RNA-Seq ( Kashima et al, 2020 ) (a time-series RNA-Seq targeting an entire embryo or tissue of each individual). Pseudo-time analysis for individual RNA-Seq might capture individual differences in the progression speed of biological events such as development.…”

Section: Introductionmentioning

confidence: 99%

Intracellular and Intercellular Gene Regulatory Network Inference From Time-Course Individual RNA-Seq

et al. 2021

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Gene regulatory network (GRN) inference is an effective approach to understand the molecular mechanisms underlying biological events. Generally, GRN inference mainly targets intracellular regulatory relationships such as transcription factors and their associated targets. In multicellular organisms, there are both intracellular and intercellular regulatory mechanisms. Thus, we hypothesize that GRNs inferred from time-course individual (whole embryo) RNA-Seq during development can reveal intercellular regulatory relationships (signaling pathways) underlying the development. Here, we conducted time-course bulk RNA-Seq of individual mouse embryos during early development, followed by pseudo-time analysis and GRN inference. The results demonstrated that GRN inference from RNA-Seq with pseudo-time can be applied for individual bulk RNA-Seq similar to scRNA-Seq. Validation using an experimental-source-based database showed that our approach could significantly infer GRN for all transcription factors in the database. Furthermore, the inferred ligand-related and receptor-related downstream genes were significantly overlapped. Thus, the inferred GRN based on whole organism could include intercellular regulatory relationships, which cannot be inferred from scRNA-Seq based only on gene expression data. Overall, inferring GRN from time-course bulk RNA-Seq is an effective approach to understand the regulatory relationships underlying biological events in multicellular organisms.

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Direct-TRI: High-throughput RNA-extracting Method for all Stages of Zebrafish Development

Cited by 9 publications

References 15 publications

Tensor Decomposition-based Unsupervised Feature Extraction Succeeded in Identification of Differentially Expressed Transcripts from Redundantde novoTranscriptome ofPlanarian

Tensor Decomposition-based Unsupervised Feature Extraction Succeeded in Identification of Differentially Expressed Transcripts from Redundantde novoTranscriptome ofPlanarian

asb5a/asb5b Double Knockout Affects Zebrafish Cardiac Contractile Function

Intracellular and Intercellular Gene Regulatory Network Inference From Time-Course Individual RNA-Seq

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