For total ILC3 RNA-Seq, live Lineage-CD127+Thy1highKLRG-1- cells were sorted from siLP of Gata3fl/fl and Gata3ΔILC3 mice by FACSAria.  Total RNA was purified by QIAzol lysis reagent and miRNeasy Micro Kit (Qiagen). 10-20 ng of total RNA was amplified with the Ovation RNA-Seq System V2 (NuGEN).   For ILC3 subsets RNA-Seq, indicated ILC3 subsets were sorted based on RORgt-E2-Crimson, Tbet-ZsGreen reporters and CCR6, NKp46 staining. Library prepare refer to the protocol above.  For ChIP-Seq, live Lineage-CD127+Thy1highKLRG-1-NK1.1- ILC3s were sorted from siLP of Rag1-/- mice, live Lineage-CD127+Thy1+KLRG-1+ ILC2s were sorted from mesenteric lymph nodes of IL-25-injected Rag1-/- mice, and Th2 cells were prepared from three round differentiated naïve CD4+ T cells as previous described. Cells were cross-linked with 1% formaldehyde for 10 min. For RNA-Seq, the double-stranded cDNA was sonicated (30” on and 30” off for 20 cycles) to an average size of 200 bp by Bioruptor Pico (Diagenode). Indexed sequencing libraries were then generated using 250 ng sonicated cDNA with the KAPA LTP Library Preparation Kit (KAPABiosystems) according to the manufacture’s protocol with minor modifications. Multiplex sequencing reads of 50 bp were generated by the NHLBI DNA Sequencing and Computational Biology Core.  For ChIP-Seq,  chromatin was prepared by sonication with Bioruptor Pico (30” on and 30” off for 9 cycles) and immunoprecipitated with anti-GATA3 (L50-823) and anti-mouse IgG-coated magnetic beads using iDeal ChIP-Seq Kit for transcription factors (Diagenode). ChIPed DNA was made into an indexed library and sequenced as described above for RNA-Seq.
GEO Accession
GSM1829643