alias: E-MTAB-6308:Cultured TEC1_p
title: Illumina HiSeq 4000 sequencing; An integrated approach to profile lung tumor endothelial cell heterogeneity across species and models and to identify angiogenic candidates
design: An integrated approach to profile lung tumor endothelial cell heterogeneity across species and models and to identify angiogenic candidates
library_construction_protocol: Freshly-dissected tissue was placed into a C-tube (Miltenyi Biotec) containing 5 ml digestion buffer (0.1% collagenase II (Thermo Fisher Scientific), 0.25% collagenase type 4 (Worthington Biochemical), DNase I (150 U/min) in DMEM supplemented with 1x sodium pyruvate, 1x MEM NEAAs, ECGF/Heparin, antibiotic/antimycotic (2x) and 1% penicillin/streptomycin (Thermo Fisher Scientific)) and processed with the m_lung_01 program of gentleMACS (Miltenyi Biotec). The samples were then incubated in a water bath at 37°C for 30 min with manual shaking every 5 min. At the end of digestion, the samples were processed with the m_lung_02 program of gentleMACS, and the reaction was stopped by adding 10 ml of an isolation buffer containing 1x PBS, 0.1% BSA, 5 mM EDTA. Subsequently, the cell suspension was filtered through a 100 m cell strainer (Corning) on ice. Finally, ECs were enriched by magnetic bead sorting using MACS system (Miltenyi Biotec) at RT. Immune cells were depleted using selection with CD45 MicroBeads (Miltenyi Biotec), followed by positive selection of ECs with CD31 MicroBeads (Miltenyi Biotec), according to the manufacturer's procedures. ​ The single cell suspensions of freshly isolated TECs and NECs (MACS-bead enriched to 20-30% as described above) or cultured TECs were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics), aiming for 6,000 cells per library. Samples were processed using kits pertaining to V2 barcoding chemistry of 10x Genomics. Single samples were always processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel. For each experiment, all samples (TEC and NEC) were processed in parallel in the same thermal cycler.​
identifiers.namespace: Laboratory of Angiogenesis and Vascular Metabolism VIB-KU Leuven Center for Cancer Biology (CCB)
identifiers.id: E-MTAB-6308:Cultured TEC1_p
attributes.tag: Experimental Factor: disease
attributes.value: lung adenocarcinoma
attributes.tag: Experimental Factor: growth condition
attributes.value: in vitro