Title: Affymetrix CEL analysis. Description:
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
normalization data transformation protocol
We used Partek Genomics Suite software for probe import: Pre-background Ajustment was done for GC Content and probe sequence. We used RMA background correction and Quantile normalization. Probes were log2 transformed and probeset summarization was done with median polish.
nucleic acid labeling protocol
We used the Affymetrix Terminal Labeling and Controls kit for fragmentation and labeling as described by the manufacturer
nucleic acid extraction protocol
After cell sorting, RNA was extracted by usign the RNeasy Micro Kit (Qiagen) according to the manufacturers protocol and stored at -80C.
Adult living mouse
Isolation of lymphocytes from the lamina propria of the small intestine was performed as described previously(Sanos SL adn Diefenbach A., Methods Mol Biol. 2010;612:505-17. ). Bones were crushed with a pestle, rinsed with icecold PBS and filtered trough a cell strainer. Red cell lysis was performed. The following lymphocytes were sorted from the lamin propria of the small intestine of EomesGfg/+ RORgtCreTGg Rosa26Yfp/+ by using the markers Lineage- CD45+ Nkp46+ NK1.1+ : 1.convential NK cells (Eomes GFP+ RORgt YFP-) 2. ILC1 (Eomes GFP- RORgt YFP-) 3. exRORgt ILC3s (Eomes GFP- RORgt YFP+). Conventional NK cells from the bone marrow were sorted from Eomes Gfp/+ mice with the markers Lineage- CD45+ NK1.1+ Eomes GFP+.