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. The mice were treated one week after castration by daily i.p. with GSK126 at the dose of 100mg/Kg for 3 weeks. 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.