As our knowledge of tumor biology grows, the interest to understand the intricacies of how cells interact within the tumor can influence patient outcomes. The importance of analyzing heterogeneous cells across the tumor microenvironment has also increased. Multi-om sequencing, which permits the acquisition of single cell RNA and attack sequencing from the same cell in a paired cell fashion provides a significant advance towards this end.
However, the quality of the data obtained from these experiments is highly dependent on the quality of the experimental conditions and inputted biological material. This protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multi-om sequencing using the 10X genomics platform. This protocol is reliable using either fresh or frozen single-cell suspensions.
We provide recommendations for tissue dissociation conditions, cryo preservation of single-cell suspensions, and assessment of isolated nuclei. In this protocol, we use human pancreatic ductal adenocarcinoma specimens, which are a primary tissue type of focus in our laboratory. Pancreas cancer represents a highly desmoplastic tumor type, which pretends relatively sticky tissue as well as cells.
Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of the cells captured. Begin by transferring previously-thawed pancreatic tumor cell suspension to a two-milliliter micro centrifuge tube, and add PBS to reach a final volume of two milliliters. Then use a hemocytometer to evaluate the quantity and quality of the cells.
Once the cell pellet is obtained through centrifugation, using progressively smaller pipet tips, carefully decant the supernatant to minimize pellet distribution while maximizing the fluid decanted. Prepare one X cell lysis buffer, then add 100 microliters of it to 900 microliters of previously prepared cell lysis dilution buffer to obtain 0.1X cell lysis buffer. Transfer 100 microliters of chilled 0.1X cell lysis buffer to the cell pellet and gently pipet five times to ensure complete re-suspension.
After incubation on ice for three minutes, add one milliliter of chilled wash buffer to the re-suspended pellet and gently pipet five times. Following centrifugation, discard the supernatant as demonstrated and repeat this washing process two more times. Load trypan blue-stained suspension into a hemocytometer to count the number of nuclei.
Re-suspend the nuclei pellet in 1X nuclei buffer based on goal-targeted nuclei recovery. Carefully pass the re-suspended nuclei through a 40 micrometer pipet tip cell strainer, and then maintain it on ice. Now re-count the nuclei.
The nuclei obtained using this method exhibited appropriate size and shape. Occasional mild stippling of the nuclear envelope can be observed and should be monitored at the final nuclear evaluation via hemacytometer.
