GSM7157185: Vehicle, biological replicate 4, scRNAseq; Mus musculus; RNA-Seq
Single cell suspensions of neuroblastoma cells were generated according to the manufacturer's instructions (Worthington Papain Dissociation System, CAS: 9035-81-1).  Breifly, minced neuroblastoma tissue is dissociated by incubation with activated papain at 37oC followed by trituration.  Papain dissociation is then halted with ovomucoid.  A single step discontinuous density gradient separates intact cells from cell membranes, and then the pelleted cells are resuspended for sequencing with 10X Genomics kits. Library was performed according to the manufacter's instructions (10x Genomics). Single cell suspensions of neuroblastoma 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.