RNA-sequencing of ILC3 during murine EAE

To determine the unique gene expression profile of central nervous system (CNS)-associated ILC3, we performed RNA-sequencing on sort-purified ILC3 from the CNS, cervical lymph nodes (cLN) and small intestine lamina propria (SI-LP) during EAE. ILC3s (Live, CD45+, CD3epsilon-, CD5-, CD8alpha -, NK1.1-, CD11b-, CD11c-, CD19-, CD127+, CD90.2+, ROR-gamma-t(GFP)+) (0.5-1 x 10^3 per tissue/mouse) were sort-purified using from Rorc-eGFP mice at day 15 active EAE (n = 4 mice, 4 samples each CNS/cLN, 2 samples SI-LP). Cells were sorted using FACSAria II cell sorter (BD Biosciences). Sorted cells were used to prepare RNA sequencing libraries by the Epigenomics Core at Weill Cornell Medicine, using the Clontech SMARTer UltraLow Input RNA Kit V4 (Clontech Laboratories). Sequencing was performed on an Illumina HiSeq 4000, yielding 50-bp single-end reads. Raw sequencing reads were demultiplexed with Illumina CASAVA (v.1.8.2). Adapters were trimmed from reads using FLEXBAR (v.2.4) and reads were aligned to the NCBI GRCm38/mm10 mouse genome using the STAR aligner (v.2.3.0) with default settings. Reads per gene were counted using Rsubread. For mouse cell-surface staining: CD45 (30-F11), CD3-epsilon (145-2C11), CD5 (53-7.3), CD8-alpha (53-6.7), NK1.1 (PK136), CD11b (M1/70), CD11c (N418), CD19 (eBio1D3), CD127 (A7R34), CD90.2 (Thy1.2) (30-H12).