GSM6895314: TKO-Replicate2; Mus musculus; RNA-Seq
Prostate tumor was minced and dissociated with mouse dissociation kit (Miltenyi Biote,130-096-730) at 37℃ for 1h. Dissociated tumor single cell suspension were passed through 40μm strainer followed by red blood cell lysis.  Then the dead cell was removed using EasySep™ Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, 17899) followed by hashtag staining and labelling. Cells from different samples were counted and combined into one samples. The cells are ready for scRNA-seq Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics, user guide CG000315). 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.