GSM6421538: ABM-AM-7 [Cpp]; Mus musculus; RNA-Seq
Neutrophils were mobilized to lung of 8-10 weeks old male Catchup (heterozygous) or non-labelled C57BL/6-background mice by exposure to 1mg/ml of nebulized LPS (Sigma, Escherichia coli O55:B5) inside the fumigation chamber. 20 mg of anti-Ly6G-BV421 antibody (Biolegend, #127628) was i.v. injected 2 minutes before sacrifice. Lungs were perfused with 10 ml of PBS through right ventricle of heart and collected for PMN isolation, biochemical, and histopathological analyses. For PMN isolation, a single cell suspension was prepared by digestion of minced lung lobes with collagenase A (1 mg/ml; Millipore-Sigma # SCR103) at a 370C shaking water bath for 45 min. Digested lung samples were passed through 16-17G needle and filtered through 40 mm cell strainer (Thermo Fisher, #08-771-1). Red blood cells were removed by using RBC lysis buffer (Thermo Fisher, #00-4333-57). A single cell suspension prepared from Catchup (heterozygous) mice were directly subjected to flow sorting for separation of td-Tomato-positive or double td-Tomato and anti-Ly6G-BV421-positive PMN. PMN, labelled with td-Tomato (or anti-Ly6G-AF-594) and td-Tomato and anti-Ly6G-BV-421 (or anti-Ly6G-AF-594 and anti-Ly6G-BV-421), were sorted in a sterile environment by MoFlo Astrios and MoFlo Astrios EQ (Beckman Coulter) cell sorter equipped with 405, 488, 561 and 635 nm lasers, and collected in RPMI-1640 medium (Thermo Fisher #72400047). Both the sorters are Gates were initially established on forward and side scatter, based on granularity and relative size. Aggregates were gated out accordingly. Selective gates were drawn to sort the single positive and dual positive PMN. Software SUMMIT, 6.1 (Beckman Coulter) was used for sorting and analysis. RNA quality and quantity was assessed using the Agilent bio-analyzer. All samples showed RNA integrity number > 8. Strand-specific RNA-SEQ libraries were prepared using a TruSEQ mRNA RNA-SEQ library protocol (Illumina provided). Library quality and quantity was assessed using the Agilent bio-analyzer and libraries were sequenced using an Illumina NovaSEQ6000 (Illumina provided reagents and protocols). TruSEQ mRNA RNA-SEQ library protocol (Illumina provided)