GSM7876515: RPM_10 week tumor_rep1; Mus musculus; RNA-Seq
A single 10-week RPM tumor was extracted from the mouse prostate, and EGFP signal was used to microdissect tumor tissue (Nikon, SMZ18). Tumor sample was sliced into ~5 mm x 5 mm pieces, dabbed on a kimwipe to remove moisture, and immediately flash frozen in liquid nitrogen. Tumor piece was loaded onto a Singulator 100 (S2 Genomics) cartridge supplemented with 3.5 μL of 1 M DTT (Sigma, 43816-10mL) and 87.5 units of Protector RNAse inhibitor (Sigma, 3335402001). Nuclei were isolated with standard-nuclei isolation protocol according to manufacturer recommendations. Nuclei suspension was cleaned with sucrose density gradient (Sigma, NUC201-1KT) in protein low-bind microcentrifuge tubes (Eppendorf, 0030108442) at 500g for 5 min. Nuclei were subsequently resuspended in nuclei wash buffer: 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1% BSA, 1 U/μL Protector RNAse inhibitor, strained through blue-capped 35 μm FACS tube, stained with 7-AAD, and flow sorted for 7-AAD+ population to obtain single nuclei suspension. Sorted nuclei were submitted to the Single Cell Analytics Innovation Lab (SAIL) at MSKCC. Nuclei were then validated for nuclear integrity under phase-contrast microscopy. Nuclei were processed on a Chromium instrument (10X Genomics) following user guide manual for 3' v3.1 snRNAseq. Nuclei were captured in droplets, emulsions were broken, and cDNA purified using Dynabeads (Thermo, 37012D) followed by PCR amplification per manual instruction. ~10,000 cells were targeted for each sample. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 - 28 cycles, i7 - 8 cycles, R2 - 90 cycles) at the Integrated Genomics Operation (MSKCC).