GSM8214797: PyMT sgIl1a-CAS9 untreated tumor (gene expression library); Mus musculus; RNA-Seq
Tumors from mice were harvested 14 days after implantation. Tumors were first minced using scissors and then digested in 25 ml glass bottles with a magnetic stirrer in 5-10 ml of DMEM with Collagenase A at 1 mg/ml (Sigma-Aldrich) and DNase I at 50 U/ml (Roche Life Sci) for 45 min in a 36° C water bath. Cell suspension was then passed through 100 μm cell strainer. Each of the tumor cell suspensions per group were then stained with anti-CD45.2-APC (Biolegend; Clone 104; Cat.No 109814) including the respective barcoded TotalSeq™-B anti-mouse Hashtag antibody in sorting buffer (DPBS +2%FBS) for 30 min on ice. Cell suspensions were then washed twice with sorting buffer before resuspended in sorting buffer including DAPI (1 μg/ml) as a viability stain. Live CD45+ cells were then sorted on a BD FACSAria for each sample, washed twice with resuspension buffer DPBS (+0.04% sterile filtered BSA) and resuspended in 30-50 μl resuspension buffer. TotalSeq™ hashtag antibodies From Biolegend Inc (San Deiego, CA, USA) were used in the experiments to multiplex samples, increase sample throughput, and improve the identification of multiples. The cell viability and counts were obtained by AO/PI dual fluorescent staining and visualization on the Nexcelom Cellometer K2 (Nexcelom Bioscience LLC, Lawrence, MA). Cells were then loaded onto the 10X Genomics Chromium Single Cell Controller at a concentration of 3,000 cells/µl in order to encapsulate 10000 cells per sample. Single cells, reagents, and 10X Genomics gel beads were encapsulated into individual nanoliter-sized Gelbeads in Emulsion (GEMs), and reverse transcription of poly-adenylated mRNA was performed inside each droplet at 53°C. The scRNA-Seq libraries were prepared following the protocol provided by the 10X genomics. The cDNA libraries were completed in a single bulk reaction using the 10X Genomics Chromium NextGEM Single Cell 3' Reagent Kit v3.1.  50,000 sequencing reads per cell for scRNA-seq were generated on the Illumina NovaSeq6000 instrument.