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RNA-seq of Human neck of femur (NOF) fracture hip and osteoarthritic (OA) cartilage

ABSTRACT: RNA-seq of Human neck of femur (NOF) fracture hip and osteoarthritic (OA) cartilage

PROVIDER: PRJNA436709 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6321830_1.fastq.gz	Fastqsanger.gz
	SRR6321830_2.fastq.gz	Fastqsanger.gz
	SRR6321831_1.fastq.gz	Fastqsanger.gz
	SRR6321831_2.fastq.gz	Fastqsanger.gz
	SRR6321832_1.fastq.gz	Fastqsanger.gz

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Project description:Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes. Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals. Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy. Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling Four sets of sample comparisons (39 comparisons in total) were made in this study. These comprised 10 OA-CTL female, 10 OA-CTL male, 10 OA-OP female and 9 OP-CTL female comparisons. A Compugen Human 19K-oligo library spotted onto glass slides by the Adelaide Microarray facility (AMF) was used in this study. The slides were interrogated by competitive hybridisation of Cy3 and Cy5 labelled pairs of OA-CTL, OA-OP or OP-CTL amplified RNA samples. Sample pairs were age-matched as closely as possible. A biological dye-swap strategy was employed. After hybridisation and washing of the slides they were scanned using a GenePix 4000B Scanner driven by GenePix Pro 4.0. All analyses were performed using the statistical programming and graphics environment R. The “SPOT” software package was used to identify spots by adaptive segmentation method and subtract backgrounds utilising morphological opening approach. Data analysis was performed in R using Bioconductor. Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin. Linear modelling was performed with the Limma package of Bioconductor. Linear models were used to incorporate all available data into a single analysis. This allowed the use of both direct and indirect comparisons in calculation of expression ratios as well as improved accuracy in estimation of variance for each gene. Factorial design utilised the following factors: medical condition (OA or OP or CTL); sex (male or female) for OA patients only.

Project description:To reveal the organisation of the cartilage cell chondrocyte genome and identify changes that occur within this organisation during development and due to osteoarthritis (OA). Methods Assay for Transposase -Accessible Chromatin using Sequencing (ATAC-seq) was performed on chondrocytes isolated from 16 patients undergoing total hip replacement because of OA (n=7) or due to a neck of femur fracture (NOF, n=9). ATAC-seq was similarly performed on bone-marrow mesenchymal stem cells (BM-MSC) and differentiated chondrocytes of two donors. DNA sequence reads (av. 50 million/sample) were aligned to human genome Hg38. Peaks were called using MACS2 and differential accessibility identified by DiffBind. Interexperiment comparisons and intersection with published gene expression changes, chondrogenesis ChIP-seq, knee ATAC-seq and human tissue scATAC-seq were performed in Galaxy and R. OA GWAS signal regions were overlapped with our defined chondrocyte ATAC-seq peaks. Results In BM-MSC and derived chondrocytes we mapped 138005 open chromatin regions, of which 20979 and 50699 significantly increased and decreased respectively during cell differentiation. In hip chondrocytes we identified 115295 open chromatin regions, 1383 and 573 were more or less differentially accessible respectively when comparing OA with NOF samples. In both data sets ‘newly accessible regions were enriched at enhancer regions (defined by ChIP-seq). Comparing the data with the ATAC-seq from the single cell ATLAS we identified 11866 open regions exclusive to chondrocytes. Genes associated with these regions were significantly enriched for cartilage-related gene ontology terms. Taking the 420 OA GWAS signals present in the GWAS catalogue, 313 of the OA regions (defined as lead SNP + proxy SNPs with r2 ≥ 0.8) overlapped with a chondrocyte ATAC-seq region. Conclusions Here we have mapped chromatin accessible region changes during chondrogenesis, showing that newly accessible regions are enriched at enhancer regions and positively correlate with gene expression. Open chromatin region changes between OA and NOF cartilage were fewer, and peak differences were subtle. Overall, we have associated OA GWAS loci with accessible regions and defined regions of the genome specific to cartilage and chondrogenesis.

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RNA-seq of Human neck of femur (NOF) fracture hip and osteoarthritic (OA) cartilage

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