GSM6876524: Tumor patient 8 (WDLPS), scRNAseq; Homo sapiens; RNA-Seq
Fresh tumor samples were cut in small pieces then dissociated 30 min at 37C in CO2-independent medium (Gibco) containing 150 mg/ml of Liberase TL (Roche) and DNase 1 (Sigma Aldrich). Dissociated cells were then filtered with a cell strainer and washed in PBS. Debris were removed by centrifugation (3,000 xg for 10 min at 4C), using the debris removal solution (Miltenyi Biotech). Cells were then resuspended in PBS, counted and adjusted at 10⁶ cells/ml. 6,000 cells were loaded on a 10X Chromium (10X Genomics) and libraries were prepared using a Single Cell 30 Reagent Kit (NextGem kit, 10X Genomics), according to the manufacturer's protocol, targeting 3,000 recovered cells per cell type and sample. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing platform. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.