GSM6588152: scRNA,SD24h; Mus musculus; RNA-Seq
Each mouse was anesthetized with pentobarbital (i.p. 80 mg/kg) and taken 500 μL blood retro-orbitally which was collected in an EDTA anticoagulant tube (BD Biosciences). The blood sample was transferred into 5-fold volume of Ammonium-Chloride-Potassium (ACK) lysis buffer (150 mM NH4Cl, 10mM KHCO3, Na2·EDTA 0.1nM), and incubated on ice (gently turn upside down 2 times during incubation) for 10 min. After 500×g centrifugation at 4℃ for 10 min, the supernatant was completely discarded. The cell pellet was washed with 2-fold volume of ACK lysis buffer, and 500×g centrifuged at 4℃ for 10 min. The leukocyte pellets of 3 mice in the same group were resuspended in equal amount with DPBS (containing 0.04% BSA), and adjusted the cell concentration to 500-1,200 cells/μL. Each library was designed to contain around 7,000 cells, and sequencing depth was about 50,000 reads per cell. All libraries for single-cell RNA sequencing were prepared by Chromium Single Cell 3' Reagent Kits (v3 Chemistry) (10x Genomics) according to the manufacturer's instructions. Qualitative analysis of each DNA library was performed using an Agilent 2100 Bioanalyzer. The concentration of each DNA library was measured using a Qubit fluorometer (Invitrogen).