|
| Status |
Public on Aug 15, 2017 |
| Title |
POVA [251911910877_S01_2_3] |
| Sample type |
RNA |
| |
|
| Source name |
CD4 T cells (cDCs+OVA)
|
| Organism |
Mus musculus |
| Characteristics |
origin: CD4 T cells after coculture with cDCs in the presence of OVA
cell type: Primary CD4 T cells
|
| Treatment protocol |
T cells were cocultured with the corresponding subset of DCs (8:1 T cell/DC ratio) in the presence or absence of chicken ovalbumin (OVA) 323-339 peptide for 18 h. CD4+ T cells from the coculture were sorted on a flow cytometer.
|
| Growth protocol |
Mouse naive CD4+ T cells were isolated from cell suspensions of lymph nodes or spleen. Cell suspensions were incubated with biotinylated antibodies (BD Biosciences) against CD8, CD19, CD25, CD11b, CD11c, CD45R, MHC-II (I-Ab), DX5, IgM, Gr-1 and F4/80 and subsequently with streptavidin microbeads (MACS; Miltenyi Biotec). CD4+ T cells were negatively selected in auto-MACS Pro Separator (Miltenyi Biotec) according to the manufacturer's instructions. At this point DC preparations were characterized as CD11c+ MHCII+ Ly6G-. To obtain both pDCs and cDCs, bone marrow cell suspensions were cultured with 100 ng ml-1 Flt3-ligand for 8 d and maturation was subsecuently induced by 6 µg ml-1 of CpG. pDCs and cDCs were isolated using MACS system B220-labeled beads and auto-MACS Pro Separator (Miltenyi Biotec) being pDCs B220+ fraction and cDCs B220- fraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Quiazol reagent and the miRNeasy® mini kit (Quiagen),
|
| Label |
Cy3
|
| Label protocol |
miRNA Labeling Kit (Agilent Technologies) was used to label RNA. Basically, 100 ng of total RNA were dephosphorylated and Cyanine 3-pCp molecule was ligated to the 3´ end of each RNA molecule by using T4 RNA ligase.
|
| |
|
| Hybridization protocol |
100 ng of Cy3 labelled RNA were hybridized for 20 hours at 55ºC in a hybridization oven (G2545A, Agilent) set to 15 rpm in a final concentration of 1X GE Blocking Agent and 1X Hi-RPM Hybridization Buffer, according to manufacturer's instructions (miRNA Microarray System Protocol, Agilent Technologies).
|
| Scan protocol |
Arrays were scanned at 5mm resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies) using the default settings for miRNA Microarray v2.0 (miRNA Microarray System Protocol, Agilent Technologies).
|
| Description |
Expression of miRNAs from CD4 T cells sorter from coculture with cDCs + OVA
|
| Data processing |
Images provided by the scanner were analyzed using Agilent´s software Feature Extraction version 10.7.3.1 and protocol miRNA_105_Jan09
|
| |
|
| Submission date |
Aug 09, 2016 |
| Last update date |
Aug 15, 2017 |
| Contact name |
Fatima Sanchez-Cabo |
| E-mail(s) |
fscabo@cnic.es
|
| Phone |
+34 91 4531200
|
| Organization name |
CNIC
|
| Street address |
Melchor Fernandez Almagro
|
| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8824 |
| Series (1) |
| GSE85363 |
microRNA profiles of CD4 T cells after cognate antigen specfic stimulation |
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