|
| Status |
Public on Dec 25, 2015 |
| Title |
Sheep red blood cells, Effector T cells_003 |
| Sample type |
RNA |
| |
|
| Source name |
Mice injected with 2x10^9 sheep red blood cells i.p.
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
gender: female
tissue: spleen
sorting parameters: Effector T cells: CD19-, CD4+, CXCR5-, PD-1+
|
| Treatment protocol |
Mice were immunised with sheep red blood cells (2x10^9 i.p.) or S. enterica (10^6 colony forming units of SL3261 strain, i.v.). Mice were left untreated for 6 days, at which point spleens were harvested for analysis.
Red blood cells were lysed with RBC lysis buffer. Splenocytes were enriched in CD4+ T cells by negative selection with Dynabeads (Invitrogen). Cells were then stained for flow cytometry and sorted over BD Aria flow cytometer sorter based on the expression of surface markers as described in the table.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by passing TRIZOL over Qiagen Rneasy columns as per manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA sample integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies). 100-300ng of total RNA was used for cRNA synthesis and fragmented. Labeling was performed with One-Cycle cDNA synthesis Kit (Affymetrix) according to manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
cRNA was hybridized to Affymetrix Mouse Gene 1.1 ST Arrays according to manufacturer’s protocol.
|
| Scan protocol |
Arrays were scanned with a GeneChip Scanner 3000 (Affymetrix) according to manufacturer’s protocol.
|
| Data processing |
Data were processed using rma and limma packages in R/Bioconductor. Pairwise group comparisons were undertaken using linear modelling. Subsequently, empirical Bayesian analysis was applied, including vertical (within a given comparison) p value adjustment for multiple testing, which controls for false discovery rate, using the limma Bioconductor package
The normalised data are log2 values for the probes reliably detected on all arrays, processed using rma followed by quantile normalisation.
|
| |
|
| Submission date |
Nov 04, 2014 |
| Last update date |
Dec 25, 2015 |
| Contact name |
Alasdair Ivens |
| E-mail(s) |
al.ivens@ed.ac.uk
|
| Phone |
44 131 6513605
|
| Organization name |
Centre for Immunity, Infection and Evolution
|
| Street address |
Kings Buildings
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17997 |
| Series (1) |
| GSE62961 |
Investigating heterogeneity within T follicular helper cells (TFH) population. |
|
