title: GSM7698164: Treated1; Mus musculus; RNA-Seq
library_construction_protocol: Tumors were harvested together, minced with a scalpel blade, and placed immediately into pre-warmed digestion media containing RPMI media, 2mg/mL collagenase IV, 0.5mg/mL collagenase II, and 20 IU/mL DNAse I. The tumor samples were incubated at 37oC for 30mins on an orbital shaker. Dissociated cells were sequentially strained to remove clumps and pelleted at 500G. To remove red blood cells, 5mL of 1X RBC Lysis Buffer was added to each sample and incubated at room temperature for 10mins. Cells were washed with 1X PBS and 0.5% BSA, centrifuged at 500G and then resuspended in 15mL of RPMI with 5% FBS. The mixture was then gently transferred on to 30mL Ficol Percoll cushion and centrifuged at 1600G for 30mins at 4oC with both slow acceleration and deceleration. Live cells present at the interface were collected and washed twice with PBS and 0.5% BSA. Cell viability and counting was performed with a Countess II and trypan blue staining. Cells were centrifuged once more and resuspended in PBS with 1% BSA at a final concentration of 1000 cells/μL. A total of 10,000 cells per tumor samples were submitted for single cell capture. The 10x Chromium v2 Single Cell platform was used to create uniquely barcoded single-cell gel beads in emulsion. Libraries were prepared using the Chromium Single Cell 3' Library and Reagent Kit v2.