Gene Expression
Nebulas
Gene Expression NebulasSummary: To understand the interplay between cancer and stroma, we performed single cell RNA-sequencing of PDAC cells admixed with stromal fibroblasts and defined different single cell populations with varying levels of proliferative and metastatic transcriptional states. PDAC cell behavior in vitro and in vivo on these phenotypic axes could be tuned with the proportion of stromal fibroblasts. These cell types were identified in human pancreatic tumors, and specific subpopulations were associated with worsened outcomes.
Overall Design: 92 single PDAC cells and 92 single CAF cells were micromanipulated and prepared for sequencing (23 of each cell type from four culture ratios). The 24th sample from each cell type-culture condition combination is a population control obtained by micromanipulating 100 cells of the given type from the given culture condition and preparing it as if it were a single cell, giving a total of 96 PDAC samples and 96 CAF samples. During the course of library construction, 3 samples were lost, all PDAC cells from the 30:70 condition (two single cells and the population control), leaving 93 total PDAC samples and 96 total CAF samples.
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| Growth Protocol: | PDAC cells and CAFs were seeded in 6-well plates in 5 different ratios: 100% PDAC, 50:50 PDAC:CAF, 30:70 PDAC:CAF, 10:90 PDAC:CAF, and 100% CAF. DMEM (high glucose, pyruvate) with 10% fetal bovine serum (FBS) and 100 U/mL Penicillin and 100 mg/mL Streptomycin was used for all cultures. |
| Treatment Protocol: | - |
| Extract Protocol: | After 72 hours of culture, cells were trypsinized and resuspended in PBS solution, and micromanipulated. PDAC cells or CAFs were then individually micromanipulated using a 10 m transfer tip on an Eppendorf TransferMan NK2 micromanipulator, transferred into PCR tubes containing RNA protective lysis buffer (Clontech Laboratories), and flash frozen in liquid nitrogen. |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| 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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