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Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

ABSTRACT: Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

PROVIDER: PRJNA673480 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12955136.fastq.gz	Fastqsanger.gz
	SRR12955137.fastq.gz	Fastqsanger.gz
	SRR12955138.fastq.gz	Fastqsanger.gz
	SRR12955139.fastq.gz	Fastqsanger.gz
	SRR12955140.fastq.gz	Fastqsanger.gz

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Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma.

Project description:BackgroundThe purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders.MethodsWe performed single-cell RNA sequencing on paired skin biopsy specimens from 3 patients with LS. Each patient with one sample cryopreserved in CryoStor® CS10 and one fresh in RPMI media using 10× Genomics sequencing.ResultsLevels of cell viability and yield were comparable between CryoStor® CS10 (frozen) and RPMI (fresh) preserved cells. Furthermore, gene expression between preservation methods was collectively significantly correlated and conserved across all 18 identified cell cluster populations.ConclusionComparable cell population and transcript expression yields between CryoStor® CS10 and RPMI preserved cells support the utilization of cryopreserved skin tissue in single-cell analysis. This suggests that employing standardized cryopreservation protocols for the skin tissue will help facilitate multi-site collaborations looking to identify mechanisms of disease in disorders characterized by cutaneous pathology.

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Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

Project description:In this study, we compare paired fresh (RPMI) compared to frozen (Cryostor®CS10) frozen skin biopsies evaluated by 10X Genomics single cell RNA sequencing. Comparisons of the cell viability and yield, and transcript expression and yield between the two methodologies were proven to be equivalent.

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Effect of autologous fat grafting on Localized Scleroderma (morphea)

Project description:Localized scleroderma (LoS), or morphea, refers to a group of rare autoimmune connective tissue diseases. Autologous fat grafting was able to correct volume loss in patients with LoS to improve facial disfigurement.However, whether it could exert a positive effect on reversing skin sclerosis remains unclear.

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DNA methylation patterns in juvenile systemic sclerosis and localized scleroderma

Project description:Scleroderma refers to a group of chronic fibrotic immune-mediated diseases of unknown etiology. Characterizing epigenetic changes in childhood-onset scleroderma, systemic sclerosis or localized scleroderma, has not been previously performed. The aim of this study was to assess DNA methylation differences and similarities between juvenile systemic sclerosis (jSSc) and juvenile localized scleroderma (jLS) compared to matched healthy controls. Genome-wide DNA methylation changes in peripheral blood mononuclear cell samples were assessed using the MethylationEPIC array followed by bioinformatic analysis and limited functional assessment. We identified a total of 105 and 144 differentially methylated sites compared to healthy controls in jSSc and jLS, respectively. The majority of differentially methylated sites and genes represented were unique to either jSSc or jLS suggesting a different underlying epigenetic pattern in both diseases. Among shared differentially methylated genes, methylation levels in a CpG site in FGFR2 can distinguish between LS and healthy PBMCs with a high accuracy. Canonical pathway analysis revealed that inflammatory pathways were enriched in genes differentially methylated in jSSc, including STAT3, NF-κB, and IL-15 pathways. In contrast, the HIPPO signaling pathway was enriched in jLS. Our data also suggest a potential role for NOTCH3 in both jSSc and jLS, and revealed a number of transcription factors unique to each of the two diseases. In summary, our data revealed important insights into jSSc and jLS and suggest a potentially novel epigenetic diagnostic biomarker for LS.

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Single Cell Transcriptome Conservation in Cryopreserved Cells and Tissues

Project description:Single Cell Transcriptome Conservation in Cryopreserved Cells and Tissues

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Transcriptomic evaluation of pediatric localized scleroderma skin with histological and clinical correlation

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Project description:In this study, we explored the initial results of RNA transcriptomic expression between pediatric localized scleroderma skin tissue and healthy control skin, with a subanalyses of transcriptomic expression in different disease states. LS gene signatures compared to healthy controls showed a distinct expression of an inflammatory response gene signature (IRGS) composed of IFNγ, IFNα and TNFα associated genes.

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Project description:Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. The microRNA (miRNA) isolation from human skin tissue was performed using miRNeasy FFPE kit (Qiagen). For PCR array, miRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of miRNAs from 3 normal skins, 3 SSc, 3 LSc or 3 keloid were prepared, and miRNA expression profile in each disease in vivo was evaluated using RT2 Profiler PCR Array. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into 96-well RT2 miRNA PCR Array that includes primer pairs for 88 human miRNAs (SABiosciences). Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. Control donors were each matched with diseases for age, sex, and biopsy site.

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Effect of autologous fat grafting on Localized Scleroderma (morphea)

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