GSM4859465: Cardiac mac from TAC HSC rep2; Mus musculus; RNA-Seq
The biventricles excised from mice were mechanically minced. The tissue from 1 heart was then incubated in 1 ml of DMEM containing 450 U/ml of collagenase I (Sigma-Aldrich), 60 U/ml of hyaluronidase (Sigma-Aldrich), and 60 U/ml of DNase-I (Sigma-Aldrich) for 45 min at 37 °C. The cells in the suspension were vortexed for 20 sec, filtered through a 40 μm cell strainer, and washed by 12 ml cold HBSS supplemented with 2% FBS and 0.2% BSA. The cells were then washed with PBS, and resuspended in FACS buffer. Cd45+Cd11b+Cd64+Ly6c-Ly6g- cells were separated as cardiac macrophages by FACS Aria III. Peripheral blood was collected by retro-orbital bleeding into tubes coated with K3-EDTA (Greiner). Cd45+Cd11b+Ly6c+Ly6g- cells were separated as circulating monocytes by FACS Aria III. Sorted cells were lysed in Buffer RLT plus (QIAGEN), and RNA was purified using RNeasy micro Kit (QIAGEN). Poly-A mRNA was extracted from total RNA by using an Oligo-dT beads of NEBNext Poly(A) RNA Magnetic Isolation Module (NEB), after which RNA-seq libraries were prepared using a NEBNext Ultra II RNA Library Preo Kit for Illumina (NEB) according to the manufacturer's protocol.