Dataset Information

Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

ABSTRACT: Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries. This protocol can be adapted for transcriptome analysis of oocytes from other species and be used to generate high-quality data from single embryos. For complete details on the use and execution of this protocol, please refer to Biase and Kimble (2018).

SUBMITTER: Biase FH

PROVIDER: S-EPMC8536786 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

Biase Fernando H FH

STAR protocols 20211019 4

Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries. This protocol can be adapted for transcriptome analysis of oocytes from other ...[more]

PMID: 34723212

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Project description:Skeletal muscle from meat-producing livestock such as cattle is a major source of food for humans. To improve skeletal muscle growth efficiency or quality in cattle, it is necessary to understand the genetic and physiological mechanisms that govern skeletal muscle composition, development, and growth. Satellite cells are the myogenic progenitor cells in postnatal skeletal muscle. In this study we analyzed the composition of bovine satellite cells with single-cell RNA sequencing (scRNA-seq). We isolated satellite cells from a 2-week-old male calf, cultured them in growth medium for a week, and performed scRNA-seq using the 10x Genomics platform. Deep sequencing of two scRNA-seq libraries constructed from cultured bovine satellite cells yielded 860 million reads. Cell calling analyses revealed that these reads were sequenced from 19,096 individual cells. Clustering analyses indicated that these reads represented 15 cell clusters that differed in gene expression profile. Based on the enriched expression of markers of satellite cells (PAX7 and PAX3), markers of myoblasts (MYOD1, MYF5), and markers of differentiated myoblasts or myocytes (MYOG), three clusters were determined to be satellite cells, two clusters myoblasts, and two clusters myocytes. Gene ontology and trajectory inference analyses indicated that cells in these myogenic clusters differed in proliferation rate and differentiation stage. Two of the remaining clusters were enriched with PDGFRA, a marker of fibro-adipogenic (FAP) cells, the progenitor cells for intramuscular fat, and are therefore considered to be FAP cells. Gene ontology analyses indicated active lipogenesis in one of these two clusters. The identity of the remaining six clusters could not be defined. Overall, the results of this study support the hypothesis that bovine satellite cells are composed of subpopulations that differ in transcriptional and myogenic state. The results of this study also support the hypothesis that intramuscular fat in cattle originates from fibro-adipogenic cells.

Dataset Information

Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

Publications

Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

Similar Datasets

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