Unstimulated as well as IL-18 generated NK cells were sorted by FACS (FACSAria). Unstimulated NK cells were sorted for NK1.1 positive, CD3 negative, CD11b positive and c-kit negative, alive cells. IL-18 cultured NK cells were sorted for NK1.1 positive, CD3 negative, CD11b negative and c-kit positive, alive cells.
Growth protocol
NK1.1 isolated NK cells (NK1.1 isolation Kit, Miltenyi) were cultured in RPMI + GlutaMax (Invitrogen) with 10% FCS (Sigma-Aldrich), 1% Pen/Strep (Invitrogen), 1% Pyruvate (PAA), 1% NEAA (PAA), 300 IU IL-2 (R&D), and 25 ng/ml IL-18 (MBL) over night.
Extracted molecule
total RNA
Extraction protocol
Column based (Qiagen)
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix) according to the WT Terminal Labeling Protocol (P/N 702808 Rev.1, Affymetrix)
Hybridization protocol
Samples were hybridized using Hybridization Kit materials (Affymetrix) according to the WT Terminal Labeling Protocol (P/N 702808 Rev.1, Affymetrix); Fluidics Protocol FS450_0007
Scan protocol
Affymetrix Gene ChIP Scanner 3000 7G
Data processing
Data processing was done using GeneSpring GX 11 software (Agilent). ExonRMA16 summarization was applied to core probe set intensities. probe_group_file: MoGene-1_0-st-v1.r4.pgf meta-probeset_file: MoGene-1_0-st-v1.r4.mps
