title: NextSeq 500 sequencing; Single cell sequencing analysis of senescence in Mus musculus prostate cancer models
design: Single cell sequencing analysis of senescence in Mus musculus prostate cancer models
library_construction_protocol: Prostate tumors resected from Ptenpc-/- and Ptenpc-/-; Timp1-/- mice (all three lobes, AP, DLP and VP) and were processed for single cell suspension. Minced tissue was digested by resuspending in 2 ml of Digestion Buffer composed by RPMI 10%FBS+1% Penicillin and Streptomycin, and 500 uL of Collagenase D (1 mg/mL), 50µL of DNAse (100 U/mL) and 125µL of HEPES (25 mM). The cell suspension was incubated for 1 hour at 37oC on a rocker. Then, the digestion was stopped adding 1mL of RPMI 10%FBS+1% Penicillin and Streptomycin. Then FACS staining was performed using the following antibodies: anti-Epcam-FITC and APC anti-CD45 and the cells were sorted in three different tubes: Epcam+, CD45+ and Epcam-/CD45- double negative. After counting, cells were mixed together in ratio 1:1:1 to perform single cell sequencing. There is no treatment in the samples The procedure is the one of Chromium Single Cell 3' v3.1. The cells are lysed directly in the 10x Genomic instrument Single-cell transcriptomes was performed using 10X Chromium single cell platform (Chromium Single Cell 3' v3.1) (10X Genomics, Pleasanton, CA. USA) Single-cell transcriptomes was performed using 10X Chromium single cell platform (Chromium Single Cell 3' v3.1) (10X Genomics, Pleasanton, CA. USA)