Gene Expression
Nebulas
Gene Expression NebulasSummary: To increase our understanding of psoriasis, we utilized RNA-seq to assay the transcriptomes of lesional psoriatic and normal skin. We sequenced polyadenylated RNA-derived cDNAs from 92 psoriatic and 82 normal punch biopsies, generating an average of ~38 million single-end 80-bp reads per sample. Comparison of 42 samples* examined by both RNA-seq and microarray [GSE13355] revealed marked differences in sensitivity, with transcripts identified only by RNA-seq having much lower expression than those also identified by microarray. RNA-seq identified many more differentially expressed transcripts enriched in immune system processes. Weighted gene co-expression network analysis (WGCNA) revealed multiple modules of coordinately expressed epidermal differentiation genes, overlapping significantly with genes regulated by the long non-coding RNA TINCR, its target gene, staufen-1 (STAU1), the p63 target gene ZNF750, and its target KLF4. Other coordinately expressed modules were enriched for lymphoid and/or myeloid signature transcripts and genes induced by IL-17 in keratinocytes. Dermally-expressed genes were significantly down-regulated in psoriatic biopsies, most likely due to expansion of the epidermal compartment. These results demonstrate the power of WGCNA to elucidate gene regulatory circuits in psoriasis, and emphasize the influence of tissue architecture in both differential expression and co-expression analysis.
Overall Design: 92 psoriatic and 82 normal skin samples
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | RNA was prepared from the frozen biopsies using the Qiagen Allprep kit. Skin punch biopsies were trimmed of visible subcutaneous fat and visible blood was removed by blotting on saline-soaked gauze. Biopsies were then snap frozen in liquid nitrogen and stored at -80°C. For RNA isolation, frozen skin biopsy samples were wrapped in an aluminum foil pouch pre-frozen in liquid nitrogen, placed in a prechilled steel mortar, and crushed with a hammer. The pulverized frozen tissue was quickly transferred into a 1.5-ml microcentrifuge containing 1 ml of the Allprep buffer and glass grinding beads. The tube was vigorously agitated on a Vortex mixer for 5 minutes to complete the extraction process. From this point on the sample was processed exactly as described in the kit manual. RNA quality and quantity were checked using an Agilent Bioanalyzer. |
| Library Construction Protocol: | Libraries for high throughput sequencing were prepared using the Illumina mRNA-Seq kit. Briefly, the process involved isolating polyadenylated RNA, fragmentation by heating at 90°C, random-primed cDNA preparation, ligation of adaptors, and size selection on an agarose gel. A gel slice corresponding to approximately 120 bp insert was isolated, and the DNA was extracted and amplified by 15 cycles of PCR using the adaptor primers. The gel purified final product was checked for quality and quantity on an Agilent Bioanalyzer. The library thus prepared was sequenced one sample per lane in an 8-lane flow cell on the Illumina Genome Analyzer IIx. The raw sequencing output of the 80-base single read sequencing was 2000-3000 Mb per lane corresponding to 25-37.5 million reads. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina Genome Analyzer |
| 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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