|
| Status |
Public on Mar 29, 2016 |
| Title |
Mm_S4_AdDG_B839_bssilva_IMM_JS |
| Sample type |
RNA |
| |
|
| Source name |
CD3DH γδ thymocytes
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: CD3DH (reduced TCR expression)
developmental stage: Adult stage (6 weeks)
tissue: Thymus
cell type: Lymphocyte
|
| Treatment protocol |
Concentration and purity was determined by spectrophotometry and integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA).RNA was processed for use on Affymetrix (Santa Clara, CA, USA) MoGene 2.1 ST Array by using the Affymetrix Genechip WT Pico Reagent Kit, according to the manufacturer’s protocols. Starting material: 2 ng of total RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
High Pure RNA isolation Kit (Roche)
|
| Label |
Streptavidin Phycoerythrin (SAPE)
|
| Label protocol |
20 ug of this cRNA was used for a second cycle of single-strand cDNA synthesis (Genechip WT Pico Reagent Kit; Affymetrix). 5.5 ug of single stranded cDNA was fragmented and labeled (Genechip WT Pico Reagent Kit; Affymetrix). Size distribution of the fragmented and labeled cDNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
3.5 µg of fragmented and labeled cDNA was used in a 150-µl hybridization cocktail containing added hybridization controls (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips, Affymetrix), of which 120 ul were hybridized on array strips for 20 h at 48°C. Standard post hybridization wash and double-stain protocols (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips, Affymetrix) were used on an Affymetrix GeneAtlas system, followed by scanning of the array strips.
|
| Scan protocol |
standard Affymetrix protocol
|
| Data processing |
All the microarray data analysis was done using R and several packages available from CRAN (Team 2011) and Bioconductor (Huber et al. 2015). The raw data (CEL files) were normalized and summarized with the Robust MultiArray Average method implemented in the ‘oligo’ package (Carvalho and Irizarry 2010).
|
| |
|
| Submission date |
Aug 03, 2015 |
| Last update date |
Mar 30, 2016 |
| Contact name |
Ana Rita Grosso |
| E-mail(s) |
[email protected]
|
| Organization name |
Unidade de Ciências Biomoleculares Aplicadas (UCIBIO)/Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa
|
| Department |
Departamento Ciências da Vida
|
| Lab |
Computational Multi-omics Lab
|
| Street address |
Campus de Caparica
|
| City |
Almada |
| ZIP/Postal code |
2829-516 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL17400 |
| Series (1) |
| GSE71637 |
TCR signal strength controls thymic differentiation of discrete proinflammatory γδT cell subsetsistinct TCR signal strength requirements in the thymus |
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