GSM5698694: Pten pc-/-_rep1; Mus musculus; RNA-Seq
Prostate tumors were isolated, minced and processed for single cell suspension. Tissues were digested in 2 ml of Digestion Buffer composed by RPMI 10% FBS + 1% P/S, 500 mL of Collagenase D (1 mg/mL), 50mL of DNAse (100 U/mL) and 125mL of HEPES (25 mM). The cell suspension was incubated for 50 minutes at 37°C on a rocker. Then, the digestion was stopped adding 1 mL of RPMI 10% FBS + 1% P/S. The cells suspension was filtered through a 100 mm cell strainer and kept on ice for 4 minutes. Then cells suspension was filtered again through a 40 mm cell strainer and spun down at 1500 rpm for 5 min at 4°C. FACS staining was performed using EPCAM-FITC (anti-Mo CD326, eBioscience, clone G8.8 #11-5791-82) for the murine prostate tumors. Single-cell transcriptomes was performed using 10X Chromium single cell platform (10X Genomics, Pleasanton, CA. USA). FACS-sorted Epcam+ prostate cells were used as the input source for the scRNA-seq. Cells were suspended in a phosphate buffer solution containing 0.04% weight/volume bovine serum albumin (BSA). The recommended volume of single cell suspension was loaded on a Chromium Single Cell Controller (10x Genomics) targeting ~10000 cells per murine prostate samples while for xenografts tumors we used a target cell recovery between 5000 and 10000 cells per sample. Bar- coded single-cell gel beads in emulsion (GEMs) were created by 10x Genomics! Chromium TM and then reverse transcribed to generate single-cell RNA-seq libraries using Chromium Single Cell 3' Library and Gel Bead Kit v2 (10X Genomics) according to manufacturer's instructions. Resulting short fragment libraries were checked for quality and quantity using an Agilent 2100 Bioanalyzer and Invitrogen Qubit Fluorometer. Unique molecular identifiers (UMIs), which were incorporated into the 5' end of cDNA during reverse transcription, were used to quantify the exact number of transcripts in a cell. 