11_Single nucleus sequencing
Fresh frozen tumor samples from patients with NF1 were obtained for single nucleus sequencing from the Indiana Pediatric Biobank and the IU Simon Comprehensive Cancer Center Biospecimen Repository under internal review board approved protocols. Flash frozen tumor sample were stored at -80C and shipped on dry ice to Novogene for single nucleus capture, library preparation and sequencing. Single nuclei suspensions were loaded onto Chromium Chips (10x Genomics) for 3' library preparation according to the manufacturer's recommendations. Library quality control was performed by Qubit 2.0 for preliminary determination of the library concentration, Agilent 2100 to determine the insert size, and Q-PCR to quantify the library effective concentration. The resulting library was sequenced on an Illumina sequencing platform. FASTQ files were processed with Cell Ranger software (v.7.0.0, 10X Genomics) with default parameters. Reads were aligned against the human reference assembly (hg38) with the STAR aligner. Mapped, non-PCR duplicates with valid barcodes and UMIs were filtered to generate a gene-barcode matrix for downstream analysis.