title: GSM7213434: Sample_01_healthy; Mus musculus; RNA-Seq
library_construction_protocol: Mouse prostatic tumoral tissue was dissociated to single cell suspension. Briefly, tumors were digested for 45 minutes in a Collagenase type I solution (1 mg/ml) at 37°C, 5% CO2 on a rocking platform. Cells digestion was then pelleted and incubated in 2.5% Trypsin for five minutes at 37°C, 5%CO2. Trypsin was then inactivated by complete medium with FBS and cells suspension was further passed through a syringe needle until complete dissociation and filtered through a 40 μm cell strainer. CD45+ and CD45- cells were sorted by flow cytometry, resuspended in 1ml PBS plus 0.04% BSA and washed two times by centrifugation at 450 rcf for 7min. After the second wash cells were resuspended in 30 ul and counted with an automatic cell counter (Countess II, Thermo Fisher) to get a precise estimation of total number of cells recovered. CD45+ and CD45- cells of each sample were loaded into one channel of the Single Cell Chip A using the Single Cell 3' v2 single cell reagent kit (10X Genomics) for Gel bead Emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer's protocol (10X Genomics). 50 ng of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq500 Illumina sequencing platform following 10x Genomics instruction for reads generation. A sequencing depth of at least ~ 10,000 reads/cell was obtained for each sample.