GSM9431962: NF2_VS3, Naïve; Homo sapiens; RNA-Seq
Fresh surgically-removed VS tissues were placed in DMEM or saline and transported to the lab for preparing single cell suspension. Tissues were dissociated by enzymatic digestion with collagenase/dispase at 37 C on a GentleMACs dissociator. Dissociated cells were cryopreserved in 10% dimethyl sulfoxide in DMEM containing 10% FBS until processing on the 10X Genomics' Chromium Single Cell platform and sequencing on a NovaSeq 6000 System (Illumina) Library construction was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, VS tumor cells were 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 retrotranscripted to cDNA, which contains an Ilumina 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 apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.