GSM8488329: mouse_6mth_FMC_ON2; Mus musculus; RNA-Seq
De-identified human PA samples (4 female sporadic PAs and 1 female NF1-PA; clinical information in Supplemental Table 3) were obtained under an approved Human Studies Protocol as dissociated frozen tumor single cells from the St. Louis Children's Hospital Pediatric Tumor Bank. The frozen dissociated tumor single cells were thawed and resuspended in DMEM.The cells were then stained with 7-Aminoactinomycin D (7-AAD) (Fisher Scientific, A1310) for sorting using the MoFlo (Siteman Flow Cytometry Core at the Washington University in St Louis). Live cells were collected and submitted for single cell RNA sequencing (scRNA-seq).  Nf1-OPG optic nerves and meningeal duras were collected separately from ten 12-week old mice, and pooled to prepare a single-cell suspension. Optic nerves were collected and dissociated using the neural dissociation kit (Miltenyi Biotec, 130-095-942) followed by debris (Miltenyi Biotec, 130-107-677) and myelin (Miltenyi Biotec, 130-096-733) removal. Single cell supensions from meningeal duras were prepared as described above, stained with 7-AAD, and sorted using MoFlo. Live cells were collected and sent for scRNA sequencing with TCR V(D)J enrichment. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  Gene Expression libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 5' Reagent Kit (v2 Chemistry Dual Index) user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 5' Kit v2, 16 rxns (PN-1000263), Chromium Next GEM Chip K Single Cell Kit, 48 rxns (PN-1000286) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to the manufacturer protocol.  A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library.