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Status |
Public on Jan 01, 2021 |
Title |
TH17Ag_WT_rep_1 |
Sample type |
RNA |
|
|
Source name |
WT Antigen specific Th17 cell culture
|
Organism |
Mus musculus |
Characteristics |
strain background: OTII BALB/c tissue: Spleen and mesenteric lymph nodes cell type: WT Antigen specific Th17 cell culture
|
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.
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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_2_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)
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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 |
|
Relations |
Reanalyzed by |
GSM2975767 |