GSM7156208: Female Bladder tumor pool 1; Mus musculus; RNA-Seq
Tumors were cut into approximately 0.5-2mm3 samples, transferred to a gentleMACS C-tube (Miltenyi Biotec) containing 2.3 ml of cold DMEM media. Once all the tumors had been processed, 100ul of Enzyme D, 50ul of Enzyme R, and 12.5ul of Enzyme A were added to each gentleMACS C-tube. The C-tubes were attached to the gentleMACS Dissociator with heater and incubated for 30 min at 37°C.  Once the dissociation program completed, the cell suspension was passed through a pre-wetted 40um cell strainer (Falcon), washed with cold PBS and centrifuged for 5 min at 500g at 4°C. Samples were depleted of red blood cells (RBC) using ACK Lysing buffer (Gibco). Cells were centrifuged and resuspend with 5mL FACS buffer. Dead Cell Removal Kit (Miltenyi Biotec) was further used to remove dead cells. We obtained the single-cell suspension and detected live cells using a hemocytometer with trypan blue. 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.