|
| Status |
Public on Oct 30, 2015 |
| Title |
RNA-D4_CFSElo subpopulation [BN148_D4 CFSElo] |
| Sample type |
RNA |
| |
|
| Source name |
in vitro activation of naive B cell from normal blood
|
| Organism |
Homo sapiens |
| Characteristics |
subpopulation labeling: DAPI neg CFSE low
day of isolation: D4
|
| Treatment protocol |
Stimulation cocktail 1 (2,6 µg/ml Fab'2 anti-Ig, 100 ng/ml CD40 Ligand, 1 µg/ml CpG ODN 2006 and 50 U/ml IL2) for 4 days then differentiation cocktail 2 (50 U/ml IL2, 5 ng/ml IL4, 10 ng/ml IL10) for 2 days (Le Gallou et al., 2012)
|
| Growth protocol |
Naive B cells from healthy donors were purified using magnetic cell separation (Miltenyi Biotech), CFSE-labeled and cultured at 7.5.105 cells/ml for 4 days with cocktail 1. Activated B cells were then washed and further cultured at 4.105 with cocktail 2 (Le Gallou et al., 2012)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the AllPrep DNA/RNA mini kit (Qiagen) after cell-sorting of the different subpopulations (FACS Aria, BD Biosciences).
|
| Label |
biotin
|
| Label protocol |
150ng of each of the samples with RIN value >7 were converted to biotinylated cRNA with the Illumina TotalPrep-96 RNA Amplification Kit (Ambion) using a standard T7-based amplification protocol
|
| |
|
| Hybridization protocol |
Following fragmentation, 7.5 ug of cRNA were hybridized on HG-U219 arrays in 120ul volume/well. The GeneTitan Hybridization, Wash and Stain kit (Affymetrix) was used for preparing hybridization mixes and automated processing of 96-array plates on the GeneTitan Instrument (Affymetrix)
|
| Scan protocol |
HG-U219 Array Plates were scanned on the GeneTitan instrument (Affymetrix) imaging device according to manufacturer's instructions
|
| Description |
BN148_D4 CFSElo.CEL
|
| Data processing |
Raw data were normalized with the Robust Multichip Analysis (RMA) algorithm using BioConductor Affy Package
|
| |
|
| Submission date |
Aug 28, 2015 |
| Last update date |
Oct 30, 2015 |
| Contact name |
Gilles Salbert |
| E-mail(s) |
gilles.salbert@univ-rennes1.fr
|
| Phone |
+33 223 236 625
|
| Organization name |
University of Rennes 1
|
| Department |
Biology
|
| Lab |
UMR6290 CNRS
|
| Street address |
Avenue du Général Leclerc
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL13667 |
| Series (2) |
| GSE72497 |
Cell cycle-dependent reconfiguration of the DNA (hydroxy) methylome during terminal differentiation of human B cells into plasma cells [expression array] |
| GSE72498 |
Cell cycle-dependent reconfiguration of the DNA (hydroxy) methylome during terminal differentiation of human B cells into plasma cells |
|
