GSM7068699: Small prostate 1, scRNAseq; Homo sapiens; RNA-Seq
Tissue for scRNA-seq were minced and digested with 1000 U/mL Collagenase I + 1 mg/mL DNAse I + 1% antibiotic/antimycotic for 4 h while shaking at 37 °C, followed by treatment with TrypLE Express reagent as above. After cell washing and RBC lysis, cell suspensions were subjected to debris removal with Debris Removal Solution. 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.