GSM5515174: Nf2830; Mus musculus; RNA-Seq
Immediately following euthanasia, paraspinal tumors (2 or 7 mo. Dhh-Cre;Nf1fl/fl) (n=6), and age-matched wild type dorsal root ganglia (DRG) with nerve roots (n=2) were excised within 5 min. and placed into ice-cold L-15 medium (Fisher Scientific, MT-10-045-CV). Tissue was cut into 1 mm3 pieces and placed into dissociation medium in 50 mL tubes containing 20 mL L-15 medium, 10 mg Collagenase type I (Worthington Biochemical, LS004196), and 50 mg Dispase II (Sigma-Aldrich, 04942078001) at 37°C incubator for 2 hours, with 170 rpm shaking. The dissociation process was stopped by adding 30mL DMEM (Fisher Scientific, 11-965-1188) containing 10% FBS. Samples were centrifuged at 800 x g for 5 minutes at 4°C. Supernatants were removed and cell pellets were re-suspended into 50 mL of 1X PBS containing 0.1% BSA. Suspensions were sequentially filtered with 70 µm, 40 µm, and 20 µm cell strainers and centrifuged at 600 x g for 5 minutes at 4°C. Pellets were resuspended into 100 ul of 1X PBS/0.1% BSA. Trypan blue negative viable cells were counted to determine the cell concentration for droplet based single-cell RNA sequencing (scRNA-seq). Library synthesis was performed according to manufacturer's instructions to generate barcoded cDNA libraries for scRNA-seq. Ten thousand viable cells per lane from each sample (mouse or human) were loaded using the 10X Chromium platform version 2 or 3 chemistries (3') (10X Genomics). We performed 1 full-lane sequence on 2 paired-end 75-bp flow cells using an Illumina HiSeq2500. single cell RNA-seq