title: GSM8101601: TL_patient10_all cells_2; Homo sapiens; RNA-Seq
library_construction_protocol: Isolation of FACS-sorted human fetal and adult endothelial cells and of unsorted human fetal and adult endothelial and perivascular cells for single cell RNA-seq Endothelial cells were isolated from human fetal and adult tissues using tissue digestion and subsequent CD31 + / CD45- FACS sorting whereas human endothelial and perivascular cells (all cells) were isolated from the unsorted fraction. Briefly, both fetal and adult tissues were quickly minced in a petri dish on ice, using two surgical blades. For CD31+ / CD45- FACS sorting, a cell suspension was obtained upon digesting the tissue in 2 mg/ml Dispase II (D4693, Sigma- Aldrich, Steinheim, Germany), 2 mg/ml Collagenase IV (#1710401, Thermo Fisher Scientific, Zurich, Switzerland) and 2 mM CaCl2 PBS solution for 40 min at 37°C with occasional shaking. The suspension was filtered sequentially through 100/70/40 μm cell strainers (#431751, Corning, New York, USA) to remove large cell debris. Cells were then centrifuged 500 RCF for 5 min at 4°C. In case of a visible myelin pellet 5 ml 25% Bovine Serum Albumin (BSA) (ice cold) was overlayed with 5 ml of the sample, centrifuged at 2000 RCF for 20 min (4°C). The supernatant was removed (including the lipid phase) and the pellet was resuspended in 9 ml PBS, followed by another round of centrifugation at 500 RCF for 5 min (4°C). Supernatant was subsequently discarded, while the cell pellets were resuspended in 3 ml of ACK hemolytic buffer at room temperature for 3 minutes. To stop the reaction, 30 ml of ice- cold PBS was added to the mixture and centrifuged at 500 RCF for 5 min at 4⁰C. The cell pellets were resuspended in FACS buffer (PBS + 1% Bovine Serum Albumin), a volume is taken for unsorted scRNA-seq analysis. For CD31+ / CD45 - FACS sorting, the cells were stained with anti-CD31 PE conjugated antibody in a concentration of 1:20 (#566125, clone MBC78.2, BD Pharmingen) and anti-CD45 APC conjugated antibody in a concentration of 1:20 (#17-0459- 42, clone HI30, eBiosciences) for 30 min at 4⁰ C, protected from light. Thereafter the cells were washed with 1ml of FACS buffer, centrifuged in a tabletop centrifuge at 500 RCF at 4⁰C for 5 min. Finally, the cell pellets were resuspended in appropriate volumes of FACS buffer (PBS + 1% Bovine Serum Albumin) and the suspension was passed through a 35 μm cell strainer of a FACS sorting tube (#352235, Corning). Immediately before sorting, SYTOXTM blue was added in 1:1000 (Thermo Fisher Scientific, #S34857) to exclude dead cells from further analysis. Cell debris were excluded via a forward scatter-area/side scatter-area (FSC-A/SSC-A) gating, while singlets were selected for using a forward scatter-area (FSC-A)/ FSC-height (FSC-H) gating strategy. Viable (SYTOXTM blue negative) endothelial cells were FACS-sorted by endothelial marker CD31 positivity and negative selection for the brain microglia and macrophages marker CD45, whereas unsorted endothelial and perivascular cells were obtained from the SYTOXTM blue- fraction. Cells were sorted by a FACS Aria III (BD Bioscience) sorter using the four-way purity sorting mode directly in EGM2 medium (#CC-3162, Lonza, Basel, Switzerland). Library construction was performed according to the 10x Chromium Single Cell 3' Reagent Kit v2.