NextSeq 500 sequencing; Single Cell RNA-seq from Prostate Cancer Patient Derived Xenografts and LNCaP Cells
Single Cell RNA-seq from Prostate Cancer Patient Derived Xenografts and LNCaP Cells
PDX tumors were maintained by subcutaneous implantation of matrigel-embedded tumor fragment (1-2-mmm average diameter tumor) or take rate varied from 1 to 6 months. Cells were suspended in PBS and 50% Matrigel and subcutaneously injected into the dorsal flanks of the mice (2 10^6 cells/mouse). Tumor growth was recorded using a digital caliper, and tumor volumes were calculated using the formula (L x W 2) /2, where L=length and W=width of the tumor. Tumor volume was measured 2 times per week. When the tumor reached the dimension of 50-100 mm2, mice were surgically castrated. PDX tumor tissue was cut into small pieces (1–0.5 mm) with a scalpel blade and then digested in Collagenase Type I media solution at 37 °C for 45-60 min. After enzymatic dissociation, the cell suspension was passed through a 100 µM cell strainer to eliminate macroscopic tissue pieces and  centrifuged. The cell pellet was then resuspended in 2-volume pf RBC lysis buffer, incubated for 3 min at RT, and, after centrifugation, resuspended in media. Cell suspension of PDX cells was then propagated in 3D vitro culture. The cells were embedded in 50% phenol red-free Matrigel and plated as a drop in a 96-well-plate (10000 cells/ well) and maintained in the medium 7-10 days. Samples were collected either 80 days after castration (residual)  or 120 days after castration (regrowth), and subjected to the molecular assessment. After resuspension in PBS, single-cell suspensions were loaded into a 10x Chromium Controller (10x Genomics, Pleasanton, CA, USA), aiming for 10000-5000 cells, with the Chromiun Next GEM Single Cell 3' v3.1 reagent kit (PN-1000121 10x Genomics), according to the manufacturer's instructions. Libraries construction was performed following  the Chromiun Next GEM Single Cell 3' (v3.1) reagent kit (PN-1000121 10x Genomics), according to the manufacturer's instructions.