GSM3868568: Microglia_Sample_3417_R1; Mus musculus; RNA-Seq
The testis, epididymis and brian were cut in small pieces and enzymatically digested by a solution containing RPMI1640 (ThermoFisher) + 1 mg/mL Collagenase II (Sigma) + 50μg/mL DNase I (Sigma) for 40 min at 37oC. Then they were passed through 70 μm cell strainers and the generated single cell suspensions undergone erythrocyte lysis on ice. Single cell suspensions from  testes and epididymides were pooled in a separate tube. Cells from brains, epididiymides, and testes were resuspended into 40% isotonic Percoll (GE Healthcare, Uppsala, Sweden) at a density of 1.072 g/ml in PBS containing 3% FCS. Cells were collected from 1.06 and 1.072 g/ml layers after centrifugation and resuspended. RBCs were lysed in RBC lysis buffer (Qiagen). The cells were blocked with FC-block and stained with antibodies against CD45, Ly6c, CD11b, CD11c, CD11b and F4/80. Macrophage populations were identified and sorted as live (DAPI-), CD45+ Ly6c- CD11c-, CD11b+ F4/80+. The sorted cells were collected in RPMI medium containing 10% FCS. According to the manufacturer´s instructions in the Tru-Seq RNA library preparation kit v2, 10 ng of RNA was converted into cDNA libraries
GEO Accession
GSM3868568