Dead Cell Removal from Dissociated Tumor for Single-Cell Sequencing

To begin, obtain the digested tumor tissue sample and lice the red blood cells. Centrifuge the sample at 450G for five minutes at four degrees Celsius. After removing the media, resuspend the pellet in one milliliter of the isolation media.

After repeating the centrifugation, resuspend the pellet in 100 microliters of isolation media. Transfer 100 microliters of sample into a well of a 0.2 milliliter PCR strip tube. In a biosafety hood, add 10 microliters each of annexin V cocktail and biotin selection cocktail to the sample.

Pipette mix the solution and incubate at room temperature for four minutes. Next, vortex the dextran-coated magnetic particles for 30 seconds. Mix 20 microliters of the beads and 60 microliters of isolation media with the sample.

After three minutes, place the strip tube onto a 10x Genomics magnetic separator on the high setting for five minutes at room temperature. Then transfer the liquid from the strip tube without disturbing the beads into a new 1.5 milliliter low bind micro centrifuge tube. Centrifuge the tube as demonstrated earlier, and resuspend the pellet in an appropriate volume of our PMI media based on the pellet size.

Finally, count the cells in a hemocytometer. Following dead cell removal, the live cell count was 4.9 times 10 to the power of 5 per milliliter, with viability above 90%