title: GSM7675891: Microglia-EAE-BMS202-5 [TCTGCA]; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with cold 1× PBS and the CNS was isolated and mechanically diced using sterile razors. Cortices were transferred into 5 ml of enzyme digestion solution consisting of 35.5 µl papain suspension (Worthington, #LS003126) diluted in enzyme stock solution (ESS) and equilibrated to 37 °C. ESS consisted of 10 ml 10× EBSS (Sigma-Aldrich, #E7510), 2.4 ml 30% D(+)-glucose (Sigma-Aldrich, #G8769), 5.2 ml 1 M NaHCO3 (VWR, #AAJ62495-AP), 200 µl 500 mM EDTA (Thermo Fisher Scientific, #15575020), and 168.2 ml ddH2O, filter-sterilized through a 0.22-µm filter. Samples were shaken at 80 rpm for 30–40 min at 37 °C. Enzymatic digestion was stopped with 1 ml of 10× hi ovomucoid inhibitor solution and 20 µl 0.4% DNase (Worthington, #LS002007) diluted in 10 ml inhibitor stock solution (ISS). 10× hi ovomucoid inhibitor stock solution contained 300 mg BSA (Sigma-Aldrich, #A8806) and 300 mg ovomucoid trypsin inhibitor (Worthington, #LS003086) diluted in 10 ml 1× PBS and filter sterilized using a 0.22-µm filter. ISS contained 50 ml 10× EBSS (Sigma-Aldrich, #E7510), 6 ml 30% D(+)-glucose (Sigma-Aldrich, #G8769), and 13 ml 1 M NaHCO3 (VWR, #AAJ62495-AP) diluted in 170.4 ml ddH2O and filter-sterilized through a 0.22-µm filter. Tissue was mechanically dissociated using a 5-ml serological pipette and filtered through a 70-µm cell strainer (Fisher Scientific, #22363548) into a fresh 50-ml conical tube. Tissue was centrifuged at 600g for 5 min and resuspended in 10 ml of 30% Percoll solution (9 ml Percoll (GE Healthcare Biosciences, #17-5445-01), 3 ml 10× PBS, 18 ml ddH2O). Percoll suspension was centrifuged at 600g for 25 min with no breaks. Supernatant was discarded and the cell pellet was washed once with 1× PBS, centrifuged at 500g for 5 min and prepared for downstream applications. Astrocytes were FACS-isolated using a negative gating strategy. Microglia were FACS-sorted by CD45hiCD11b+ staining. Library preparation was performed by Rupert Oellinger