NextSeq 500 sequencing; RNA-seq analysis of lung-infiltrating neutrophils from WT and Atgl-cKO mice
RNA-seq analysis of lung-infiltrating neutrophils from WT and Atgl-cKO mice
At day 10 post treatment, the mice were euthanized and Ly6G+ neutrophils were isolated from lung by using anti-Ly6G MicroBead Kit (Miltenyi Biotec) following manufacturer's instructions. The isolated neutrophils were analyzed by flow cytometry and the cells with a > 95% purity were used for the next procedure. AT3-g-csf cells (5x10^5) were injected into the fourth mammary fat pads of female WT and Atgl-cKO mice (10-week-old, n = 4/group). Total RNA was isolated from neutrophils using the miRNeasy Mini kit (Qiagen). Stranded libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems): isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. Libraries were checked for quality and concentration using the DNA 1000 assay (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturers' instructions.