Eosinophil RNA-Seq Analysis from Lung

Eosinophils were sort-purified as Sytox blue^- CD45⁺ Lineage (CD3, CD5, CD19, Ly6G)^-CD11b⁺Siglec F⁺ from the lung of Nmur1^Cre-T2A-GFPId2^flox/flox and control mice on day 7 after papain administration. Cells were sorted into RLT Plus Lysis Buffer supplemented with 40 mM DTT and RNA was isolated using the RNeasy Plus Micro kit (Qiagen) according to the protocol provided by the manufacturer. RNA-seq libraries were prepared by the MDC BIMSB Core Bioinformatic Facility using the Ovation® SoLo RNA-Seq Systems (Tecan Genomics) or theSMARTer Stranded Total RNA-Seq Kit – Pico (Takara). Sequencing was performed on a NovaSeq 6000 (Illumina), yielding 100 bp single-end reads. RNA-Seq reads were mapped to the mouse genome (GRCm38.p5) with STAR^{35 (link)} version 2.7.3a using default parameters. Reads were assigned to genes with FeatureCounts^{36 (link)} with the following parameters: -t exon -g gene_id, gene annotation - Gencode GRCm38/M12. The differential expression was carried out with DESeq2 version 1.22.1^{37 (link)} using default parameters. We kept genes that have at least 5 reads in at least 3 samples.