|
| Status |
Public on Jan 01, 2021 |
| Title |
CD4_WT_rep_1 |
| Sample type |
RNA |
| |
|
| Source name |
WT CD4+ Tcells
|
| Organism |
Mus musculus |
| Characteristics |
strain background: wild type BALB/c
tissue: Spleen and mesenteric lymph nodes
cell type: WT CD4+ Tcells
|
| Treatment protocol |
Spleen and mesenteric lymph nodes were collected and incubated with mouse biotin conjugated antibodies against IgM, B220, CD19, MHCII, CD11c, CD11b, CD44, CD8a, Dx5a (Pharmingen, BD). Naive CD4+ T cells were purified by negative selection using Streptavidin Microbeads (Miltenyi Biotec) and LD columns (Miltenyi Biotec) as indicated by manufacturer. Cells were cultured with TGFbeta and IL-6 and in the presence of OVA for the agTH17 cultures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
MicroRNA were extracted with miRNeasy minikit (Qiagen) as recommended by the manufacturer's. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
100ng of total RNA from each sample was dephosphorylated with Calf Intestinal Alkaline Phosphatase (CIP) at 37ºC for 30’ following a denaturalization and then a ligation for 2h at 16ºC using Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies; Palo Alto, CA). In this step a molecule of Cyanine3-pCp is incorporated to the 3’ end of RNA molecules.
|
| |
|
| Hybridization protocol |
Labeled RNA was dried and resuspended with Hybridization Buffer and Blocking Agent, incubated 5 min at 100ºC and transferred to an ice water bath for 5 min. Samples were hybridized in a volume of 45ul to the Mouse miRNA Microarray, 8x15K (Agilent) for 20 hours at 55°C and 20rpm. Microarrays were then washed at room temperature for 5 min in Gene Expression Wash Buffer 1 and 5 min at 37ºC in Gene Expression Wash Buffer 2 (Agilent Technologies; Palo Alto, CA).
|
| Scan protocol |
Arrays were scanned on an Agilent G2565A microarray scanner under default settings recommended by Agilent Technologies for miRNA microarrays with 100% PMT and 5μm resolution. Data was extracted using Agilent Feature Extraction software 10.7.3.1 (for raw files with Grid_Name = 019119_D_F_20091030) or 10.1.1.1 (for raw files with Grid_Name = 019119_D_20081121).
|
| Description |
S02_1_1
|
| Data processing |
Data files were then imported into GeneSpring® GX software version 10. (Agilent Technologies) and quantile normalization was performed. Probes were flagged (Present, Marginal, Absent) using GeneSpring® default settings and filtered for further analysis according to expression (all miRNAs which raw expression levels are within the range 20%-100% of expression in 75% replicates of at least one condition were retained) and flags (all miRNAs flagged as Present or Marginal in 75% replicates of at least one condition were retained). To determine statistically significant differences between both conditions, moderated t-test using the limma package from Bioconductor was applied to the filtered data. Benjamini-Hochberg p-value correction was used for control of the False Discovery Rate (FDR or Corrected p-value)
|
| |
|
| Submission date |
Feb 01, 2018 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Manuel J Gomez |
| E-mail(s) |
mjgomezr@cnic.es
|
| Organization name |
CNIC
|
| Lab |
Bioinformatics Unit
|
| Street address |
Melchor Fernández Almagro, 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8824 |
| Series (2) |
| GSE109992 |
Characterization of microRNA signature in activated CD4+ T, polyclonal Th17 (Th17 poly), OVA-specific Th17 (Th17 Ag-sp) cell skewed cultures |
| GSE109995 |
microRNA signature of murine CD4+ T cell subpopulations under different skewing conditions |
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