GSM2498348: Neutrophils from PBS livers-5; Mus musculus; RNA-Seq
Mice were anesthetized and perfused livers were collected, cut into small fragments, and incubated with 5 mL digestion buffer composed by 5% Fetal Bovine Serum (FBS, Biological Industries, Israel), 0.5 mg/ml Collagenase VIII from Clostridium histolyticum (Sigma-Aldrich, USA), 0.1 mg/ml Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, USA) in Dulbecco’s Phosphate Buffered Saline with Calcium and Magnesium (PBS+/+, Biological Industries, Israel), in a shaker-incubator at 250 rpm, 37°C for 45 minutes. The samples were then subjected to three cycles of washing with PBS-/- at 400 rpm, 4°C for 5 minutes from which the supernatant was kept, omitting the parenchymal cell pellet. Subsequently, the supernatant was centrifuged at 1400 rpm, 4°C for 5 minutes and the cell pellet was lysed for erythrocytes by 2 minutes incubation with ACK buffer composed by 0.15 M NH4Cl, 0.01 M KHCO3, and washed with PBS-/-. For RNA-seq 50,000 neutrophil cells per liver were sorted by FACSAria directly into a 1.7 ml micro-tube containing 50 μl lysis buffer (RNase-free H2O, 0.2% Triton-X (Roth) and 0.4 U/μl RNasin (Promega). Antibodies used throughout the research are: CD11b (clone M1/70), Ly6C (clone HK1.4), Ly6G (clone 1A8),and CD45 (clone 30F11). RNA-seq library production, sample preparation and analysis were carried out as previously described (Jaitin, D. A et al., Science 2014), using a method developed at Dr. Ido Amit's lab, Weizmann Institute of Science. 3' RNA-seq for digital gene expression quantitation