Illumina HiSeq 4000 sequencing; An integrated approach to profile lung tumor endothelial cell heterogeneity across species and models and to identify angiogenic candidates
An integrated approach to profile lung tumor endothelial cell heterogeneity across species and models and to identify angiogenic candidates
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. Human tumor and normal ECs were resuspended in 6 ml of a 1:1 mix of M199 (containing 10% FBS and sodium pyruvate, MEM NEAAs, MEM vitamin solution, glutamine and heparin as above) and EGM2 medium, further supplemented with antibiotic/antimycotic (2x) (Thermo Fisher Scientific), and the single cell suspension was plated out in 3 wells of a 6-well plate pre-coated with 0.1% gelatin. The next day, the medium was changed to EGM2 supplemented with antibiotic/antimycotic and thereafter changed every other day. When reaching confluency and upon detection of EC cell colonies (patches with cobblestone appearance), ECs were purified using anti-CD31 coated magnetic beads (MACS Technology, Milteny Biotec). The resulting MACS-purified ECs were further cultured in EGM2 medium and used at passage 2-5 for bulk RNA-sequencing. RNA was extracted from cultured human TECs and NECs using TRIzol (Thermo Fisher Scientific). Starting from 1μg total RNA, poly-adenylated fragments were isolated, reverse transcribed and converted into indexed sequencing libraries. Libraries were constructed using the KAPA stranded mRNA-seq kit (Sopachem).
Experimental Factor: sampling site
neoplasm