title: GSM9231556: Immune cells, RPMI-treated, 7 days post injection; Mus musculus; RNA-Seq
library_construction_protocol: Mice were anesthetized with isoflurane and underwent cardiovascular perfusion with PBS, followed by isolation of the brain. The brain was homogenized by mechanical force on 100-μm strainer. The suspension was pelleted by centrifugation at 300 x g for 5 min and resuspended in digestion buffer [1.5mg/ml Collagenase D, 10U/ml DNase I in RPMI], subsequently transferred to a gentleMACS C tube. Enzymatic digestion was performed under stirring at 160 rpm, 37 °C for 30 min. Cells were resuspended in 5ml of 30% Percoll diluted in RPMI and 2ml of PBS was carefully added on top of the layer. Immune cells were purified by density gradient centrifugation at 500 x g (with lowest acceleration and low brake) for 20 min at 18 °C. After the myelin layer and the supernatant were removed, pelleted immune cells were washed and resuspended in DPBS. Libraries were prepared using the Chromium Next GEM Single Cell 5' Kit v2 (dual index) (10X Genomics) following manufacturer's instructions.