GSM8594551: Human Colon Cancer DTCs (Rep4), GEX; Homo sapiens; RNA-Seq
We used DTCs that were obtained from the same tumor samples as our FFPE blocks to obtain TCR profiling data on three samples. Cryovials were thawed in a water bath for 2–3 minutes at 37°C with gentle shaking. Thawed cells were transferred into a 15 mL centrifuge tube containing 9 mL of pre-warmed complete growth medium (RPMI + 10% FBS) and the centrifuged at 150 rcf for 10 minutes at room temperature using a swinging bucket rotor. All but ~200 μL of the supernatant was removed and then the pellet was resuspended in this remaining 200 μL, followed by the addition of 200 μL of complete media. Cells were strained using a 40 μm Flowmi cell strained and transferred to a 1.5 mL Eppendorf tube before being counted on a Cellaca MX using AOPI. 5 μL of CD3 BV510 stain and 2.5uL of CD45 APC was added to the cell suspension and it was incubated on ice for 30 minutes in the dark. Cells were then centrifuged at 300 rcf for 10 minutes at 4°C. The supernatant was removed without disturbing the cell pellet and 1 mL of chilled PBS + 10% FBS added to the tube with gentle mixing. Cells were centrifuged at 300 rcf for 10 minutes at 4°C and the pellet resuspended in 250 μL of chilled PBS + 10% FBS. Cells were then sorted on a SONY MA900 cell sorter. Live single DTCs were gated using forward scatter (relative size) and back scatter (relative granularity) and 7AAD for live/dead cell discrimination. CD45 and CD3 were used to identify T cells. Sorts were performed on a 100 µm nozzle at 20 psi sheath pressure. The sample pressure was set to low-medium with a consistent event rate maintained throughout the sort. Cells were sorted into 30 μL 10% FBS in PBS, in a pre-coated 1.5 mL Eppendorf tube. Sorted cells were directly loaded onto the 10x chip for generation of 5′ gene expression and TCR libraries following the Chromium Next GEM Single Cell 5′ Reagent Kits v2 (Dual Index) User Guide (CG000331) on the Chromium X instrument.