GSM9594692: Prostate, uninfencted, sample 2; Mus musculus; RNA-Seq
Tissue harvest and preparation for single-cell sequencing: 28 DPI mouse prostates were harvested en bloc and placed immediately into ice-cold PBS31. Under a dissecting microscope, individual lobes were isolated as close to the urethra as possible and minced into ~1 mm² pieces in Dulbecco's Modified Eagle Medium (DMEM). Minced tissue was incubated in 5 mL of DMEM containing collagenase type IV (final concentration: 5000 U/mL) at 37 °C with rotation for 45 min. To maximize viability of cells through the digestion process, cells were collected by centrifugation every 15 min, in which buffer was collected, centrifuged at 500 × g for 5 min at 4 °C, and the resulting pellet was resuspended in 1 mL DMEM supplemented with 1% GlutaMAX and 10% fetal bovine serum (FBS), then held on ice; this mid digestion harvest was implemented to improved cell viability. The remaining tissue fragments were gently triturated five times using a wide-bore pipette tip, replenished with warm digestion medium, and returned to 37 °C. After 45 min of digestion, the supernatant was again collected, pelleted, and processed as above. Residual tissue was treated with 1 mL of dispase (5 U/mL), triturated for 2 min, and transferred into 5 mL DMEM with 1% GlutaMAX and 10% FBS. Cells were pelleted (500 × g, 5 min, 4 °C), resuspended in 1 mL of ice-cold red blood cell (RBC) lysis buffer (Roche), gently triturated, and incubated for 2 min on ice. The suspension was filtered through a pre-wetted 40 µm cell strainer and rinsed with DMEM containing 1% GlutaMAX and 10% FBS. Cells were again centrifuged (500 × g, 5 min, 4 °C) and resuspended in 100 µL of microbeads for dead cell removal (Dead Cell Removal Kit, Miltenyi Biotec). Manufacturer's instructions were followed: briefly, cells were incubated with microbeads for 15 min at room temperature, applied to magnetic LS columns, and viable cells were collected from the flow-through eluted into PBS. The resulting single-cell suspensions were diluted in DMEM with 1% GlutaMAX and 10% FBS to a final target concentration of 400–800 live cells/µL and a total of ~16,000 cells were processed to create the gel bead-in-emulsion (GEM) utilizing the 10X Genomics Chromium GEM-X Technology. The average time from prostate harvest to GEM generation and cell lysis was ~2.5 h. Libraries were prepared using the 10x Genomics Chromium platform according to the manufacturer's instructions and sequenced on an Illuminal Novaseq 6000. Raw sequencing data were processed using Cell Ranger (v7.0.1, 10x Genomics). Briefly, raw base call (BCL) files generated by the Illumina Novaseq 6000 platform were demultiplexed into sample-specific FASTQ files using Cell Ranger mkfastq. Reads were then aligned to the mm10 reference genome (GRCm38, Ensembl version M36) using the STAR aligner as implemented within Cell Ranger33. The Cell Ranger count pipeline was used to trace aligned reads back to individual cell barcodes, collapse unique molecular identifiers (UMIs), and generate gene-by-cell expression matrices. Only barcodes passing default Cell Ranger quality filters (filtered feature–barcode matrices) were retained for downstream analysis.