title: GSM5701741: brain parenchyma from sham operated C57BL/J mice; Mus musculus; RNA-Seq
library_construction_protocol: Mice were injected (intravenously; iv) with fluorochrome coupled anti-mouse CD45 antibody (clone 30-11F, Biolegend, 3 µg/mouse). Five minutes later, animals were intracardially perfused with cold PBS with 10 U/ml heparin under deep ketamine/xylazine anesthesia. During perfusion, blood was collected and transferred into a tube. To separate different CNS compartments, the forebrain and cerebellum were dissected from mice. Under binoculars, the dura was dissected from the calvaria and the pia was dissected from the brain parenchyma. All choroid plexuses were removed from the ventricles of the brain. Brain tissue was cut into pieces and digested with collagenase D (2.5 mg/ml, Roche Diagnostics) and DNase I (0.05 mg/ml, Sigma) at 37 °C for 20 min. Pia, dura, and CP were digested in medium containing 1 mg/ml Collagenase D (Roche) for 45 min at 37°C. Afterwards, digested tissue was passed through a 70 μm cell strainer. The parenchyma was centrifuged on a 70%/37% percoll gradient. The interphase was removed, washed and re-suspended in PBS with 2% FCS. Cells extracted from the CNS, pia and dura were sorted for CD45+CD45iv- cells. Single cell suspensions were loaded onto the Chromium Single Cell Controller using the Chromium Single Cell 3' Library & Gel Bead Kit with v2 and 3 chemistry (both from 10X Genomics, #1000128, #1000157). Sample processing and library preparation was performed according to manufacturer's instructions using AMPure beads (Beckman Coulter, A63881). Sequencing was carried out on a Illumina Novaseq using NovaSeq 6000 S4 Reagent Kit (300 Cycles, #20028312) the a 150-8-8-150 read setup. scRNA-seq