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| Status |
Public on May 01, 2024 |
| Title |
MPRA in primary human macrophages |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
We investigated an intergenic haplotype on chr21q22, linked to five different inflammatory diseases. We used a functional approach (massively-parallel reporter assay; MPRA) to first identify active enhancers at the locus in primary human macrophages, and then determine if candidate variants within these regulatory regions might alter enhancer activity. In doing so, we discovered a mechanism that orchestrates macrophage responses during chronic inflammation and delineated how the risk haplotype increases expression of the causal gene, ETS2.
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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 macrophages. An oligo library was designed to test the chr21q22 candidate SNPs within the 99% credible set (based on SuSiE fine-mapping of IBD GWAS data) and to tile the region containing these candidate SNPs. 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 inflammatory (TPP) macrophages from 8 healthy individuals. Raw data are provided as Illumina reads of the 11nt barcode from mRNA extracted 24 hours after transfection, or from 4 replicates of the input MPRA vector library. We also provide tab-delimited files containing the raw barcode counts for each sample and the quantile normalized element counts (sum of barcodes tagging a single genomic sequence).
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| Contributor(s) |
Lee JC |
| Citation(s) |
38839969 |
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| Submission date |
Apr 12, 2023 |
| Last update date |
Jun 14, 2024 |
| Contact name |
James C Lee |
| E-mail(s) |
james.lee@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| 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) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
| GPL33331 |
Illumina HiSeq 2500 (Homo sapiens; synthetic construct) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA954701 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE229472_barcode_counts.txt.gz |
150.3 Kb |
(ftp)(http) |
TXT |
| GSE229472_normalised_collapsed_constructs.txt.gz |
12.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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