title: GSM6746727: cutaneous neurofibroma; Homo sapiens; RNA-Seq
library_construction_protocol: Single-cell suspensions of cNF and pNF were obtained from NF1 patient. Samples were mechanically dissociated and enzymatically digested in 0.5% collagenase type I and 2% FBS in PBS at 37℃ for 3 hours. The cell suspension was filtered and washed. Erythrocytes were removed by adding ACK Lysing Buffer to the remaining pellet. Live cells were sorted by excluding 7-aminoactinomycin D-positive cells using Cell Sorter SH800. Library was performed according to the manufacturer's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, cell suspension was resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane Dynabeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.