title: GSM8953516: Secondary-Inf-ILCs_S3; Mus musculus; RNA-Seq
library_construction_protocol: Lung tissue was dissected, mechanically dissociated, and enzymatically digested in DMEM containing Liberase, Dispase II, and DNase I at 37°C for 40 minutes with agitation. Cell suspensions were filtered, stimulated with Cell Activation Cocktail (with Brefeldin A), and stained for viability, surface, and intracellular markers using standard protocols. ILC2s (CD45⁺ CD90.2⁺ CD127⁺) were sorted by FACS with >85% viability and resuspended in PBS with 10% FBS at 700–1200 cells/μL. Single-cell RNA-seq libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagent Kit v3 per manufacturer's instructions. Next-generation sequencing libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagent Kit v3 (10x Genomics), following the manufacturer's protocol. Libraries were uniquely indexed with the Chromium Dual Index Kit, pooled, and sequenced on an Illumina NovaSeq 6000 system using a paired-end, dual-indexing run. Each library was sequenced to a target depth of 20,000 mean reads per cell.