GSM8197847: KO_minus; Mus musculus; RNA-Seq
mice were perfused with 20 ml PBS under deep anesthesia. The hearts were collected and minced with scissor into about 0.5 mm in 5 mL of RPMI-1640 with 10% Fetal Bovine Serum (FBS). Then digested with Collagenase II (200 U/mL, WOR) and DNase I (60 U/ml, WOR) a 37°C incubator for 30 min with agitation on a rotator and stirred in the gentleMACS Program m_heart_01 of the gentleMACS Dissociator. The Incubation was again carried out for 30 min and stirred in gentleMACS Program m_heart_02. The homogenate of cardiac tissue was passed through a 70 µm cell strainer, added 10 ml of wash buffer (PBS supplemented with 1% FBS) and centrifuged at 400 x g for 10 min and resuspended in PBS. Single-cell suspension was stained with anti-CD45.2, CD11b, Ly6G, SiglecF. CD45.2+ CD11b+ Ly6G+ SiglecF+ neutrophils and CD45.2+ CD11b+ Ly6G+ SiglecF- neutrophils were isolated using FACS Aria II (BD Biosciences). For each sample, neutrophils from eight animals were pooled to make the cell numbers at least 2 × 104. RNA was extracted using ISOGEN (Nippon Gene). Library was constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina, and NEBNext Poly(A) mRNA Magnetic Isolation Module (Cat# E7770 and E7490; New England Biolabs).