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| Status |
Public on Mar 21, 2020 |
| Title |
Resolving molecular mechanisms of autoimmune disease in primary CD4 T cells |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
Numerous genomic loci have been implicated in autoimmune disorders, but attempts to identify causal variants, and thereby disease mechanisms, have been hampered by strong linkage disequilibrium – leaving most loci unresolved and the potential of GWAS unfulfilled. To overcome this, we developed a massively-parallel reporter assay for use in primary CD4 T-cells, a key effector of many autoimmune diseases, and sought to resolve potential causal variants via their functional effects. This provided testable hypotheses into disease mechanisms, which can provide previously unappreciated insights into molecular mechanisms of disease
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| Overall design |
The study consists of a massively parallel reporter assay, in which the minimal promoter in the vector was replaced by the RSV promoter. This was necessary in order for the assay to work in primary CD4 T cells. A 99,990 oligo library was designed to test 264 candidate SNPs at 14 autoimmune disease-associated gene deserts, and to tile each haplotype at 50bp intervals. Allelic constructs were tiled across each SNP in triplicate, and each was tagged by 30 unique 11nt barcodes. Each tiling construct was tagged by 6 unique barcodes. Transfections were performed into resting and activated CD4 T cells from 12 healthy individuals, and resting and activated Jurkats in 4 independent replicates. Raw data are provided as Illumina reads of the 11nt barcode from RNA extracted 24 hours after transfection, or from 4 replicates of the MPRA vector library. We also provide a tab-delimited file containing the unnormalized barcode counts for each sample and metadata relating to the sequence tagged by the barcode.
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| Contributor(s) |
Lee JC |
| Citation(s) |
32239644 |
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| Submission date |
Aug 17, 2019 |
| Last update date |
Jun 20, 2020 |
| Contact name |
James C Lee |
| E-mail(s) |
james.lee@crick.ac.uk
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| Organization name |
The Francis Crick Institute
|
| Lab |
Genetic Mechanisms of Disease
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| Street address |
The Francis Crick Institute, 1 Midland Rd
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| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platforms (2) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
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| Samples (36)
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| Relations |
| BioProject |
PRJNA560653 |
| SRA |
SRP218659 |
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