GSM9055765: Mammary tissue, Luc, rep3; Rattus norvegicus; RNA-Seq
For the tissue collection, rats were euthanized at the humane endpoint using the CO2 chamber. Mammary tumors and axillary and inguinal mammary glands were minced using the scalpel and incubated in the tissue dissociation media (DMEM/F12 medium containing 2 mg/mL collagenase type IV (Worthington), 2 mg/mL hyaluronidase from bovine testes (Millipore Sigma), and 2 mg/mL BSA (Millipore Sigma)) for 1 to 2 hours at 37°C, followed by storage in the freezing media (90% FBS containing 10% DMSO) in liquid nitrogen. Samples of mammary tumors or mammary glands in frozen vials were thawed at 37°C and suspended in pre-warmed DMEM/F12 media containing 20% FBS. After washed with ice cold PBS, cells were incubated with TrypLE™ Select Enzyme (1×) (Gibco, Thermo Fisher Scientific) at 37°C for 5 minutes. After neutralized with DMEM/F12 media containing 20% FBS, cells were washed with PBS twice. At the last step of washing, cells were passed through 100 μm cell strainer. Cells were then resuspended in PBS containing 0.04% w/v UltraPure BSA (Thermo Fisher Scientific) to be 1,000 cells/μl × 40 μl. Library preparation and sequencing was conducted as a fee-service of Translational Immunogenomics Lab at DFCI. Briefly, the scRNA-seq library was prepared using 10,000 cells with the Chromium Next GEM Single Cell 5' HT v2 protocol (10x Genomics). Quality control for amplified cDNA libraries and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The pooled libraries were sequenced on Novaseq X plus platform (Illumina).