Gene Expression
Nebulas
Gene Expression NebulasSummary: Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining tissue integrity. We have previously shown that mouse skin connective tissue, the dermis, is comprised of functionally distinct fibroblast lineages. However, the extent of fibroblast heterogeneity in human skin is unknown. Here, using a combination of spatial transcriptional profiling of human and mouse dermis and single cell transcriptional profiling of human dermal fibroblasts, we show that there are at least four distinct fibroblast populations in adult human skin. We define markers permitting prospective isolation of these cells and show that although marker expression is rapidly lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signalling, T cell communication and the ability to support human epidermal reconstitution in organotypic culture. Furthermore, while some fibroblast subpopulations are spatially segregated, others are not. These findings have profound implications for normal wound healing and diseases characterized by excessive fibrosis, and suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications.
Overall Design: Spatial RNA sequencing of human papillary versus reticular dermis for 3 individuals, and single cell RNA sequencing of dermal fibroblasts for a single individual
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| Growth Protocol: | - |
| Treatment Protocol: | Spatial RNA sequencing was performed using fresh human skin samples (from three separate individuals). Skin samples were incubated with Dispase II (Stemcell Technologies) for 1h at 37℃ permitting separation of the epidermis. The dermis was separated into papillary (upper 100 μm) reticular (200-500 μm) dermis by microdissection under a dissecting microscope. For single cell RNA sequencing, single cells were isolated by flow cytometry and sorted into individual wells of a 96 well plate containing 2μl lysis buffer (0.2% (vol/vol) Triton X-100 and 2 U/ul recombinant RNase inhibitor (Clontech). |
| Extract Protocol: | Separated papillary and reticular dermis samples were transferred to lysis buffer containing 2-mercaptoethanol (PureLink RNA micro scale kit, Invitrogen) and homogenized for 2 minutes using a mechanical homogenizer (Polytron, Kinematica). Subsequent RNA extraction was performed using the PureLink micro kit according to the manufacturer' instructions. |
| Library Construction Protocol: | Library preparation was performed with SmartSeq2 followed by the Nextera XT protocol (Illumina). Sequencing was performed on the Illumina HiSeq 2000 or HiSeq 2500 with TruSeq SBS v3 chemistry. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000; Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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