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Single-Cell RNA-Sequencing Atlas of Bovine Caudal Intervertebral Discs: Discovery of Heterogeneous Cell Populations with Distinct Roles in Homeostasis

ABSTRACT: Single-Cell RNA-Sequencing Atlas of Bovine Caudal Intervertebral Discs: Discovery of Heterogeneous Cell Populations with Distinct Roles in Homeostasis

PROVIDER: PRJNA744746 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15068930_1.fastq.gz	Fastqsanger.gz
	SRR15068930_2.fastq.gz	Fastqsanger.gz
	SRR15068931_1.fastq.gz	Fastqsanger.gz
	SRR15068931_2.fastq.gz	Fastqsanger.gz
	SRR15068932_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose: Degenerative changes of the intervertebral disc are a leading cause of back pain and disability worldwide. However, precise mechanisms driving the initiation and progression of pathology have remained elusive. We find that adhesion G-protein coupled receptor Adgrg6 plays a critical role in maintaining postnatal intervertebral disc homeostasis. The goal of this study is to uncover early molecular changes in Adgrg6-defeicnet intervertebral discs prior to overt histopathology. Methods: Intervertebral disc mRNA profiles of 20-day-old wild type and Col2Cre; Adgrg6f/f mutant mice were generated by deep sequencing in triplicates. Cutadapt and perl scripts were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC. We used HISAT2 to map reads to the genome of Mus Musculus (GRCm38.88). The mapped reads of each sample were assembled using StringTie. Then, all transcriptomes from biological samples were merged to reconstruct a comprehensive transcriptome using perl scripts and gffcompare. After the ﬁnal transcriptome was generated, StringTie and Ballgown was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM. The differentially expressed mRNAs were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package Ballgown. Results: We found 884 differential expressed genes with statistical significance (p value < 0.05, and with a more stringent cut-off adjusted p value <0.05 and fold-change >2, we observed 42 differential expressed genes. Enriched pathways and biological processes using gene ontology (GO) terms included extracellular matrix, positive regulation of fibroblast proliferation, extracellular matrix structural constituent conferring tensile strength, regulation of tyrosine phosphorylation of STAT protein, and ion transport. We also find significantly increased expression of fibrotic collagens, induced expression of some Suppressor of Cytokine Signaling (SOCS) genes, and dysregulated expression of some components associated with ion transport system. Several of the significantly upregulated genes are associated with biomarkers or risk of lumbar disc degeneration and osteoarthritis in humans or animal models. Conclusions: Our study provides detailed analysis of intervertebral disc transcriptomes from both wild type and Adgrg6-deficient mice, with three biological replicates, prior to overt histopathology. Our transcriptomic analysis demonstrated a robust dysregulation of several important pathways and components of the intervertebral disc homeostasis, including induction of fibrotic gene expression, alteration of ion transport component, as well as changes in some chondrogenic and catabolic factors, prior to the onset of histopathology and disc degeneration. These data strongly suggest that ADGRG6 signaling is a critical factor for the maintenance of healthy gene expression profiles in the IVD.

Dataset Information

Single-Cell RNA-Sequencing Atlas of Bovine Caudal Intervertebral Discs: Discovery of Heterogeneous Cell Populations with Distinct Roles in Homeostasis

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