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Status |
Public on Mar 29, 2020 |
Title |
Tmem_lvempty_B |
Sample type |
RNA |
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|
Source name |
primary T lymphocytes derived from human peripheral blood mononuclear cells (PBMC)
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Organism |
Homo sapiens |
Characteristics |
cell type: memory T lymphocytes culture conditions: transduced with lv-empty, supplemented with IL-2 (50 U/ml)
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Treatment protocol |
Cells were selected for transduction with puromycin (2ug/ml, Sigma-Aldrich). Before collection, samples were treated for 3h with PMA/ionomycin.
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Growth protocol |
memory T lymphocytes were isolated by magnetic enrichment for CD4 (Miltentyi Biotec) and sorted for CD4+CD25-CD45RA-CCR7+/-. Cells were activated with plate-bound aCD3 and aCD28 for 2 days, before removed from stimuli and transduced with the indicated lentiviral constructs. Cells were cultured another 10 days and from day 5 on IL-2 (50 U/ml, provided by the institute) was added to the media.
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Extracted molecule |
total RNA |
Extraction protocol |
Memory T cells were resuspended in Trizol and total RNA was extracted with Direct-Zol RNA mini Prep (Zymo Research)
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Label |
biotin
|
Label protocol |
50ng of total RNA were labelled with the nCounter® Immunology Panel (Human V2) codeset according to the manufacturers protocol
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Hybridization protocol |
Samples were hybridized overnight at 65C
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Scan protocol |
Samples were run on a Nanostring Sprint Profiler
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Description |
Human peripheral blood mononuclear cells were separated from peripheral blood using Ficoll-Paque Plus (GE Healthcare). CD4+ T lymphocytes were further isolated by positive selection using magnetic microbeads (Miltenyi Biotec). Memory T cell subsets were then sorted based on the expression of the following surface markers: CD4+CD25–CD45RA-CCR7+-. nCounter® Immunology Panel (Human V2)
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Data processing |
Raw data was processed with nSolver 3.0 Software (Nanostring). Reads were normalized to internal postive & negative controls and to included housekeeping genes. Genes were considered expressed if the sum of reads in one of the transduction groups was >150. Differential expression and p-values were calculated within the nSolver 3.0 software
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Submission date |
Nov 26, 2018 |
Last update date |
Mar 29, 2020 |
Contact name |
Chiara Balestrieri |
E-mail(s) |
balestrieri.c@gmail.com
|
Organization name |
IRCCS San Raffaele Scientific Institute
|
Department |
Center for Omics Sciences
|
Street address |
Via Olgettina 58
|
City |
Milan |
ZIP/Postal code |
20132 |
Country |
Italy |
|
|
Platform ID |
GPL21847 |
Series (2) |
GSE122942 |
A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes (Nanostring I) |
GSE122946 |
A molecular network regulating the proinflammatory phenotype of human memory T lymphocytes |
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