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| Status |
Public on Aug 12, 2021 |
| Title |
RNASeq of Candida-stimulated CD3+ T-cells from individuals with CARD9 mutations and normal controls |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
CD3+ T cells were enriched using the EasySep human T cell isolation kit (Stem cell technology). T cells from normal controls or patients with CARD9 mutations were co-cultured with monocytes of one normal control in the presence of heat killed candida. After 3 days, T cell were enriched again to remove the monocytes and total RNA was extracted from the T cells for the RNA-sequencing (RNASeq) evaluation. Targeted RNA sequencing library preparation was carried out using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies), which profiles more than 20,000 human genes; each amplicon (~150 bp) represents a unique targeted gene (one transcript per gene). For library preparation, each sample was run in duplicate, and a cDNA library was generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified with Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified with Agilent Bioanalyzer high-sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with 4 individual samples per pool. Pooled libraries were amplified and enriched with the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies) with Ion PI HiQ kit and chip version 3. We performed gene-level differential expression analysis of targeted RNASeq data using R (v.3.5.3) and the Bioconductor packages DESeq2 (v.1.22.2).
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| Overall design |
RNA sequencing was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit and Ion Torrent Proton sequencing system (Life Technologies). We analyzed RNA samples extracted from human Candida-stimulated CD3+ T-cells from normal controls and patients with CARD9 mutations.
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| Contributor(s) |
Rosenzweig S, Kuehn H, Niemela J, Stoddard J, Goel S |
| Citation(s) |
34791587 |
| |
| Submission date |
Aug 09, 2021 |
| Last update date |
May 12, 2023 |
| Contact name |
Julie E Niemela |
| E-mail(s) |
[email protected]
|
| Organization name |
NIH Clinical Center
|
| Department |
Dept. of Lab Medicine
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| Lab |
Sequencing Lab
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| Street address |
10 Center Dr
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL17303 |
Ion Torrent Proton (Homo sapiens) |
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| Samples (11)
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| Relations |
| BioProject |
PRJNA753095 |
| SRA |
SRP331696 |
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