GSM7051063: Relapse_1; Mus musculus; RNA-Seq
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 isolated using the Anti-ACSA-2 MicroBead Kit (Miltenyi, #130-097-678) and the pellets flash frozen in liquid nitrogen. RNA isolation, and library preparation using Single Primer Isothermal Amplification (SPIA) was performed by BGI Tech Solutions. Library preparation using Single Primer Isothermal Amplification (SPIA) and library pooling was performed by BGI Tech Solutions.