|
| Status |
Public on Nov 25, 2014 |
| Title |
8 NK |
| Sample type |
RNA |
| |
|
| Source name |
Naive Tonsil
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Tonsil
stimulation: Naive/Healthy
treatment: FACS sorted
cell population: DAPI- CD3- CD19- CD14- CD94+ CD127lo/- CD56+
|
| Treatment protocol |
ILC3, NK cells and the CD34+ HPC subsets were sorted by flow cytometry from tonsils of 6 distinct donors. The ILC3 and NK cells were sorted after MACS-depletion of CD34+ cells followed by CD56+ cell magnetic enrichment as DAPI- CD3- CD19- CD14- CD94- CD127hi CD56+ ILC3 and DAPI- CD3- CD19- CD14- CD94+ CD127lo/- CD56+ NK cells, respectively. The CD34+ HPC subsets were separated after MACH enrichment of CD34+ cells according to expression of CD34 and CD117 in DAPI- CD56- CD3- CD19- CD14- CD117+ CD34dim, DAPI- CD56- CD3- CD19- CD14- CD117- CD34dim and DAPI- CD56- CD3- CD19- CD14- CD117- CD34bright cells. All cell populations were sorted to purity above 95%, pelleted and stored at -80 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the NucleoSpin® RNA kit (Macherey-Nagel)
|
| Label |
Cy3
|
| Label protocol |
10 ng of total RNA was used for the samples: 7 c+, 7 NK, 8 high, 8 c+, 9 c+, 10 c+, 10 c -, 87 c+, for all other samples the complete available amount of RNA was used as template to produce Cy3- labeled cRNA. The RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Cy3 labeled cRNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Human Genome Oligo Microarrays (8x60K v2, Design ID 039494) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with wash buffer 1 for 1 min at RT followed by a second wash with wash buffer 2 for 1 min at 37°C.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent Technologies, Palo Alto, USA).
|
| Description |
Sorted as: DAPI- CD3- CD19- CD14- CD94+ CD127lo/- CD56+
|
| Data processing |
The Agilent Feature Extraction Software (FES 10.7.3.1 ) was used to read out and process the microarray image files.
|
| |
|
| Submission date |
Nov 12, 2014 |
| Last update date |
Nov 27, 2014 |
| Contact name |
Chiara Romagnani |
| E-mail(s) |
[email protected]
|
| Phone |
+493028460681
|
| Organization name |
Deutsches Rheuma Forschungszentrum - Leibniz-Gemeinschaft
|
| Lab |
Innate Immunity
|
| Street address |
Chariteplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE63197 |
Human RORγt+ CD34+ cells are lineage-specified progenitors of group 3 RORγt+ innate lymphoid cells |
|
