Gene Expression
Nebulas
Gene Expression NebulasSummary: Next Generation Sequencing Analysis of Transcriptomes in maculopapular drug rash with COVID-19 infection
Overall Design: Purpose: Investigate whether maculopapular drug rash with COVID-19 infection (COVID19-MDR) exhibits a distinct gene expression in the skin as compared to non-COVID19-MDR. Methods:RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) skin biopsies from COVID-MDR (n=3), MDR (n=7), and Healthy control (n=5). Library preparation for RNA-seq was performed by using the TruSeq Stranded RNA library preparation kit including polyA enrichment (Illumina) from total RNA. Sequencing was performed on the the Illumina NextSeq 500 platform with 75 cycles. Results: RNA sequencing from lesional skin showed that pathways of cytolysis and eosinophil chemotaxis were activated in COVID MDR. Cytolysis/cellular defense response related genes, such as PRF1 (perforin), GZMA (Granzyme A) and GNLY (Granulysin), as well as eosinophil migration/lymphocyte chemotaxis genes, e.g. C-C Motif chemokine ligand 5 (CCL5), CCL13, were upregulated in COVID MDR. Conclusions: This study provide an opportunity to understand the pathogenesis of MDR with the COVID-19 infection. We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.
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| Growth Protocol: | All blood and skin samples were taken on the day of symptom onset, that is, prior the initiation of any treatment. Skin punch biopsies were taken from all patients for histopathological evaluation (n = 12), skin punch biopsies for research purposes were available only for patients in Zurich: COVID‐MDR (n = 4), MDR (n = 7) and DRESS (n = 4), all obtained from lesional skin on the trunk. Skin from HC was obtained as discarded tissue from cutaneous surgery (n = 5).; All blood and skin samples were taken on the day of symptom onset, that is, prior the initiation of any treatment. Skin punch biopsies were taken from all patients for histopathological evaluation (n = 12), skin punch biopsies for research purposes were available only for patients in Zurich: COVID‐MDR (n = 4), MDR (n = 7) and DRESS (n = 4), all obtained from lesional skin on the trunk. Skin from HC was obtained as discarded tissue from cutaneous surgery (n = 5). Skin samples were formalin‐fixed and paraffin‐embedded (FFPE). Blood samples were obtained COVID‐MDR, COVID w/o MDR, DRESS, MDR patients and HC (n = 5 each). Blood, collected using serum tubes, was processed immediately after collection and stored at −80°C until further processing. |
| Treatment Protocol: | Skin samples were formalin‐fixed and paraffin‐embedded (FFPE). Blood samples were obtained COVID‐MDR, COVID w/o MDR, DRESS, MDR patients and HC (n = 5 each). Blood, collected using serum tubes, was processed immediately after collection and stored at −80°C until further processing. |
| Extract Protocol: | RNA was extracted from FFPE skin biopsies of 5 HC skin samples and lesional skin biopsies of MDR (n = 7) and COVID‐MDR (n = 4) patients with a Qiagen® RNeasy FFPE Kit. |
| Library Construction Protocol: | Library preparation for RNA‐seq was performed by using the TruSeq Stranded RNA library preparation kit (Illumina) from total RNA. Sequencing was performed on the Illumina NextSeq 500 platform. One COVID‐MDR sample showed very few reads and thus had to be excluded for further analyses. For further details, see the Supplementary Materials and Methods. |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | 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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