title: GSM8101630: MET_patient2_EC_bulkRNA-seq; 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). The libraries were prepared following Illumina TruSeq stranded mRNA protocol. The quality of the RNA and final libraries was determined using an Agilent 4200 TapeStation System. The libraries were pooled equimolecularly and sequenced in an Illumina NovaSeq sequencer (single-end 100 bp) with a depth of around 20 Mio reads per sample.