Gene expression signatures of innate lymphoid cells from human blood

Innate Lymphoid cells (ILCs) are a functionally and phenotypically heterologous group of small lymphocytes, which lack conventional lineage markers but can become potent effector cells upon appropriate stimulations producing large amount of cytokines. Here we investigate three different subsets of circulating (as opposed to tissue-resident) ILCs: ILC1, ILC2 and ILC3. A 2-round sorting procedure was used to obtain highly pure ILC subsets from buffy coat from healthy donor blood. The samples (3 ILC subsets per donor and 6 donors, or 18 samples in total) were analysed individually by RNAseq. CD3, CD14 and CD19 cells were MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) depleted and then sorted using a FACS Aria flow cytometer (BD Bioscience, Allschwil, Switzerland). Lymphocytes were initially gated based on FSC and SSC, and then dead cells were excluded using a Fixable Viability Dye (eBioscience, Frankfurt, Germany). We then gated on the lineage-CD127+ viable lymphocytes population, of which the CD161 mid/+ cells were the main ILC-containing population. The three ILC subsets were finally distinguished based on their differential expression of c-kit, CRTH2 and NKp44. The sorted ILC populations were immediately re-sorted into 96-well plate at 1000 cells per well or as close as possible. The wells contained lysis buffer as per user manual, SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Clontech, Japan) and snap frozen. The sorted ILC subsets were thawed and cDNA prepared using the above Clontech kit as per user manual. Illumina Nextera Library preparation and sequencing was performed by Functional Genomic Center, University of Zurich.