GSM9363181: day1ICAM1negative_2; Mus musculus; RNA-Seq
Approximately 150 µl of blood was collected from the tail artery into a tube containing 100 µl of 50 mM EDTA-2K in PBS at 1 day after the infection. The red blood cells were lysed with ACK lysing buffer (Thermo, A1049201). Cells were fixed with DSP solution (1mg/ml in DMSO, filtered through a 20 µm mesh filter (pluriSelect)) at room temperature for 30min to inactivate virus, and the fixation step was stopped by adding 1M tris (pH7.5) for the final concentration of 20mM. Cells were incubated at 4°C for 10 min firstly with purified anti-mouse CD16/32 (1:50, clone 93, Biolegend, 101301), washed, and subsequently with the set of staining antibodies; PerCP/Cyanine5.5 anti-mouse CD45.2 (1:100, clone 104, Biolegend, 109828), APC/Cyanine7 anti-mouse CD11b (1:100, clone M1/70, Biolegend, 101226), PE anti-mouse CD182 (CXCR2) (1:100, clone SA044G4, Biolegend, 149303), APC anti-mouse CD54 (ICAM1) (1:300, clone YN1/1.7.4, Biolegend, 116120). Subsequently, cells were subjected to collected with Fluorescence-activated cell sorting (FACS) using FACS Aria III or IIIu (BD Biosciences). Total RNA was isolated from isolated cells or tissues of mice with the use of RNAiso Plus reagent (Takara). RNA libraries were prepared using SMART-Seq Stranded Kit (Takara) following manufacturer's protocols.