GSM7520052: bulkRNA_Th1_D3_KO3; Mus musculus; RNA-Seq
cell isolation protocol: Cells from spleen were obtained by mechanical disruption. Peritoneal cells were obtained by washing of the peritoneal cavity with cold PBS. Isolated cells were further sorted as based on their surface markers. in vitro culture protocol: All cells were stimulated in RPMI medium with 10% (vol/vol) FCS (Invitrogen), 2 mM glutamine (Invitrogen), 100 IU/ml penicillin (Invitrogen), 0.1 mg/ml streptomycin (Invitrogen), and 20 mM HEPES buffer, pH 7.2–7.5 (Invitrogen), and 2 mM β-mercaptoethanol (Sigma-Aldrich). NK cells were treated with 1000 U/ml IL-2 and 10 ng/ml IL-12 (R&D) for 6 hours; ILC2 cells were treated with 50 ng/ml IL-25 (BioLegend) and 50 ng/ml IL-33 (Biolegend) for 4 hours; NCR+ILC3 were treated with 50ug/ml of IL-23 (R&D Systems) for 6 hours. Total RNA was extracted from 200,000 cells using QIAzol Lysis Reagent and the Direct-zol microprep RNA kit. mRNA purification, cDNA synthesis and cDNA library preparation was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490), NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, #E7770) and NEBNext Multiplex Oligos for Illumina (NEB, #E6609). cDNA libraries were sequenced at 20 million reads per sample with 50bp paired-end reads using NovaSeq or NextSeq (Illumina).