GSM8465521: Brain, MI mice; Mus musculus; RNA-Seq
Peripheral blood was collected by retro-orbital bleeding and red blood cells were lysed in RBC lysis buffer (Biolegend). Brain and hearts were excised after perfusion with PBS (Thermo Fisher Scientific), minced and digested with 450 U/mL collagenase I, 125 U/mL collagenase XI, 60 U/mL DNase I and 60 U/mL hyaluronidase (Sigma) in PBS for 40 min at 37 °C. Brain samples were passed through a 70-μm cell strainer and mixed with 30% percoll layered on top of 70% percoll. The percoll gradient was centrifuged at 500g for 30 min with the brake off. The cell fraction was collected and washed with PBS before downstream applications. For heart and brain samples, CD45+ cells were selected with a MACS column (Miltenyi). Briefly, cells were incubated with anti-CD45 microbeads (Miltenyi Biotech) then passed through a MACS separation LD column. Viability of single cells was assessed using Acridine Orange/Propidium Iodide viability staining reagent (Nexcelom), and debris-free suspensions of >80% viability were deemed suitable for the experiments. Library was constructed according to manufacturer's protocol (10X, Single Cell 5' PE)