Single cell RNA sequencing of subcutaneous nerves from 3-month-old control and mutant mice in the context of Neurofibromatosis type 1
Single cell RNA sequencing of subcutaneous nerves from 3-month-old control and mutant mice in the context of Neurofibromatosis type 1
20 nerves per mouse were dissected and HBSS was added (Sigma, ref: H6648-500ML) on ice. After mechanical dissociation, nerves were transfered in a mix of enzymes (0,15% Trypsin (Sigma #T9201), 0,04% Hyaluronidase (Bioconcept #LS005474), 0,3% Collagenase type 2 (Bioconcept #LS004174) and DNAse I (25 μg/mL)) in HBSS, during 2 hours in a water bath. After digestion, nerves were tritured with a 1000 pipette and a solution of 20% FBS in RPMI (Gibco, ref: 42401018) was added to stop digestion. Tubes were centrifuged and supernatant removed. Single viable cells were sorted (BD InfluxTM Cell Sorter) using a nuclear labelling DRAQ7TM Far-Red Fluorescent Live-Cell Impermeant DNA Dye (Abcam, ref: ab109202). Cells were collected in 20% FBS-RPMI solution. Cells were centrifuged, the supernatant removed, and myelin depletion was performed by using anti-myelin beads (Miltenyi Biotec, ref: 130-096-733) in a 0,5% of MACS solution (Miltenyi Biotec, ref: 5130-091-376). Cells were collected after the passage through the magnetic separator. Around 25,000 cells were loaded into one channel of the Chromium system using the V3.1 single cell reagent kit (10X Genomics) to generate single-cell GEMs  Following capture and lysis, cDNA was synthesized, then amplified by PCR for 12 cycles as per the manufacturer's protocol (10X Genomics).