GSM8567435: Spleen_TJ003; Mus musculus; RNA-Seq
Tumor tissues were minced carefully using scissors 1-2 mm diameter pieces in cell staining buffer (Biolegend, Cat# 420201), and placed in digestion media (10ml HBSS, HEPES 100ul, Collagenase P 10mg, BSA 100mg, and DNase I 10mg per tumor) inside a 50ml tube and incubated on a rotator at 37°C for 25 minutes. The digestion was then stopped by adding 10 ml HBSS including 10% FBS. The cell suspension was passed through a 40μm cell strainer, and removed red blood cells by resuspending in 5 ml of ACK red blood lysis buffer (Gibco) for 5 minutes. Zombie NIR staining was performed using the Zombie NIR Fixable Viability kit (Biolegend, Cat# 423105) according to its instructions for 20 minutes at room temperature. After Zombie NIR staining, cells were incubated with TruStain FcX Plus (BioLegend, Cat# 156603) for 10 minutes on ice, followed by staining with the flow cytometry antibodies all at 1:200 dilution for 25 minutes on ice. Cells are then washed thoroughly and resuspended for flow cytometry analysis. Spleen was mashed through a 70 μm cell strainer with the plunger end of a 5 ml syringe into single cell suspension. Without digestion, spleen single-cell samples were then proceeded with red blood cell lysis and cell staining using the same protocol as tumors. The 10x Genomics Single Cell Gene Expression 3' and 5' workflows are both high-throughput, scalable technologies that encapsulate thousands of individual cells (or nuclei) in droplets for lysis and reverse transcription from up to 8 samples in parallel. The droplets contain primers for cell- and and molecule-specific barcoding of cDNA. We can prepare libraries for CITE-Seq/feature barcoding, CRISPR, or cell hashing. Using our 5' workflow, we can profile the full length V(D)J regions from T-cell or B-cell receptors from human and mouse samples. The resulting libraries are typically sequenced on an Illumina NovaSeq 6000. Users must deliver live cells (or nuclei) in suspension to the Core