Deciphering cellular and molecular signatures of malignant progression in Neurofibromatosis type 1 using single-cell transcriptomic analysis
Deciphering cellular and molecular signatures of malignant progression in Neurofibromatosis type 1 using single-cell transcriptomic analysis
Mice were euthanised via isoflurane inhalation and perfused with PBS to remove circulating immune cells. Tumors were dissected and sampled for (immuno)histological and genomic analyses. Remaining tissue was minced into a slurry and digested in RPMI-1640 medium (Gibco, #42401-018) containing 0,25 mg/ml of Liberase HD (Roche, #05401089001) for 30 min at 37°C. Enzymatic digestion was halted by adding 10% FBS in RPMI (Gibcon #10270106) and tumor cell suspensions were collected by centrifugation (1200 rpmi for 5 min)and mechanical dissociation and filtering (30µm MACS SmartStratiners, Miltenyi Biotec, #130-098-458)was performed to obtain single cell suspension  single-cell suspensions were labelled with DAPI (Invitrogen, #D1306) and subjected to FACS to recover viable cells. 20,000 of viable  were loaded into one channel of the Chromium system using the V3 and V3.1 single-cell reagent kits (10X Genomics Following capture and lysis, cDNAs were synthesized, then amplified by PCR for 12 cycles as per the manufacturer's protocol (10X Genomics).