title: GSM8377403: Prostate  Control 9 month post tam Replicate 2 Sequencing Run 2; Mus musculus; RNA-Seq
library_construction_protocol: To distinguish circulating immune cells from tissue resident immune cells, mice were injected intravenously with 3μg of anti CD45.2-FITC for 3 minutes prior to euthanasia. The dorsal-lateral-ventral lobes (DLV lobes) of mouse prostates from PTEN(i)pe-/- mice and age-matched PTENL2/L2control littermates at 3months and 9months post tamoxifen (PIN and adenocarcinoma stage respectively) were isolated and dissociated by enzymatic digestion(collagenase 2 / DNase) to generate a single-cell suspension. For surface staining, single cell suspensions were preincubated with blocking anti-Fc receptor antibody (clone 2.4G2) at 4°C for 10 min. Cells were then washed and stained with appropriate fluorescent labeled monoclonal surface antibodies (CD45, CD11b, Ly-6G) at 4oC for 30 min at dark. Prostate samples from three PTEN(i)pe-/- mice at 3 months post tamoxifen were pooled. Similarly, prostate samples from five  sex-matched PTENL2/L2 control littermates at 3 months post-tamoxifen were also pooled. The same approach was applied for the 9 months samples of PTEN(i)pe-/- and control mice respectively. This experimental procedure was repeated twice in order to generate duplicates for each timepoint and sample type to eliminate possible batch effects during the analysis. Next, samples were FACS-sorted using a BD FACSAriaTM Fusion flow cytometer. Cell viability was evaluated using DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride). The control samples were sorted as bulk resident immune cells (CD45.2-CD45+). However, for the tumorous PTEN(i)pe-/- samples, the tissue resident neutrophils (CD45.2-CD45+CD11b+Ly-6G+) were sorted independently from the rest of the immune cells (CD45.2-CD45+CD11b+Ly-6G-). Post sorting, they were re-pooled in a ratio of 30% neutrophils to 70% of rest immune cells. Trypan blue exclusion assay was used to determine cell number viability of each FACS-sorted sample using a Neubauer Chamber. Samples with a cell viability of >95% were processed with a Chromium Controller (10X Genomics, Leiden, The Netherlands). Sixteen thousand cells were loaded per well in nanoliter-scale Gel Beads-in Emulsion (GEMs). Single-cell 3' mRNA-seq libraries were generated according to “User guide Chromium Next GEM Single Cell 3 prime Reagent Kits v3.3 (Dual index) (10x Genomics, PN CG000315- Rev.D). Briefly, cells were partitioned into droplets with barcoded gel beads and reverse transcription master mix using Chromium Chip G. After complementary DNA (cDNA) synthesis and barcoding from poly-adenylated mRNA, GEMs were disrupted and pooled before amplification of cDNA by 11 polymerase chain reaction (PCR) cycles. Following enzymatic fragmentation and size selection of cDNA amplicons, sequencing libraries were constructed by adding Illumina P5 and P7 adapters (San Diego, USA) and i5 and i7 sample indexes by end repair, A-tailing, adaprot ligation, and 10 cycles of PCR amplification. Quality control and quantification of libraries was performed with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). The generated libraries were sequenced on NextSeq2000 sequencer using a P3-type flow cell and 100-type cartridge. Two runs of sequencing were performed for each libray. Next, bioinformatic analysis was followed. scRNA-seq (10x Genomics 3'v3.1)