Gene Expression
Nebulas
Gene Expression NebulasSummary: As organisms age; cells accumulate genetic and epigenetic changes that eventually lead to impaired organ function or catastrophic failure such as cancer. Here we describe a single-cell transcriptome analysis of 2544 human pancreas cells from donors; spanning six decades of life. We find that islet cells from older donors have increased levels of disorder as measured both by noise in the transcriptome and by the number of cells which display inappropriate hormone expression; revealing a transcriptional instability associated with aging. By analyzing the spectrum of somatic mutations in single cells from previously-healthy donors; we find a specific age-dependent mutational signature characterized by C to A and C to G transversions; indicators of oxidative stress; which is absent in single cells from human brain tissue or in a tumor cell line. Cells carrying a high load of such mutations also express higher levels of stress and senescence markers; including FOS; JUN; and the cytoplasmic superoxide dismutase SOD1; markers previously linked to pancreatic diseases with substantial age-dependent risk; such as type 2 diabetes mellitus and adenocarcinoma. Thus; our single-cell approach unveils gene expression changes and somatic mutations acquired in aging human tissue; and identifies molecular pathways induced by these genetic changes that could influence human disease. Also; our results demonstrate the feasibility of using single-cell RNA-seq data from primary cells to derive meaningful insights into the genetic processes that operate on aging human tissue and to determine which molecular mechanisms are coordinated with these processes.
Overall Design: Examination of single cells from primary human pancreas tissue
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Single-cells were collected in lysis buffer in 96-well plates, followed by reverse transcription with template-switch using an LNA-modified template switch oligo to generate cDNA. After 21 cycles of pre-amplification, DNA was purified and analyzed on an automated Fragment Analyzer (Advanced Analytical). |
| Library Construction Protocol: | Each cell’s cDNA fragment profile was individually inspected and only wells with successful amplification products (concentration higher than 0.06 ng/ul) and with no detectable RNA degradation were selected for final library preparation. Tagmentation assays and barcoded sequencing libraries were prepared using Nextera XT kit (FC-131-1024; Illumina) according to the manufacturer’s instructions. Barcoded libraries were pooled and subjected to 75 bp paired-end sequencing on the Illumina NextSeq instrument. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| 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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