Micrornaeasy

Freshly sorted ILC2s after three intranasal administrations of rmIL-33 (0.5 μg per mouse), defined in this study as IL-33-activated ILC2s (aILC2s), were incubated with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with CD200-Fc or the isotype control for 24 h (5 × 10⁴ mL⁻¹). Total RNA was isolated using MicroRNAeasy (Qiagen). scRNA sequencing data [GSE102299] for mouse and human samples were obtained as described before^{17 (link),44 (link)}. In total, 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system. Raw reads were aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0 Copyright; Partek Inc. Normalized read counts were tested for differential expression using Partek’s gene-specific analysis (GSA) algorithm leveraging lognormal with shrinkage^{45 (link)}. To explore expression gradient and narrow z-score range, we separately analyzed CD200R⁺ and CD200R^- ILC2s. During the analysis of CD200R⁻ ILC2s, we assigned z-scores of zero (black) to all ILC2s without any CD200R mRNA. In a second parallel analysis of CD200R⁺ ILC2s, we assigned z-scores of zero (black) to ILC2s with the lowest non-zero CD200R expression level (z-score of 5.22 in our data set) using Partek Flow.

Livers were collected at the indicated times after transcardial perfusion to clear organs of red blood cells. Liver samples were digested in collagenase IV (MP Biomedicals, LLC) at 37 °C for one hour and then processed on a 70 µm nylon cell strainer (Falcon) into a single cell suspension. Liver samples were resuspended in 30% Percoll solution and centrifuged at 750g for 20 min without brake. Supernatants were discarded and pellets were resuspended in RBC Lysis Buffer (BioLegend) for 5 min. Cells were then washed and ready for magnetic separation. The CD11c MicroBead kit UltraPure (Cat#130-125-835, Miltenyi Biotec) was then used according to the manufacturer’s conditions in order to isolate CD11c⁺ cells. Total RNA was isolated using MicroRNAeasy (Qiagen, Valencia, California) from freshly isolated hepatic CD11c⁺ cells. 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system as previously described^{63 (link)–65} . Raw reads were aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0; Partek Inc., St. Louis, MO, USA. Pathway analysis was performed using the Qiagen IPA software.

Freshly sorted ILC2s after three intranasal administrations of rm-IL-33 (0.5 µg per mouse), defined in this study as activated ILC2s (aILC2s), were incubated (5 × 10⁴ per mL) with rm-IL-2 (10 ng mL⁻¹) and rm-IL-7 (10 ng mL⁻¹) for 24 h. Total RNA was isolated using MicroRNAeasy (Qiagen). In total, 10 ng of input RNA was used to produce cDNA for downstream library preparation. Samples were sequenced on a NextSeq 500 (Illumina) system. Raw reads were aligned, normalized and further analyzed using Partek Genomics Suite software, version 7.0 Copyright; Partek Inc. Normalized read counts were tested for differential expression using Partek’s gene-specific analysis (GSA) algorithm.