MC38 tumor mass were excised and minced and digested with 0.25% Liberase TM (Merck) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) at 37 °C for 30 minutes with gentle shaking. Cells were passed through a 100-μm filter and subjected to 40% Percoll centrifugation to remove debris and epithelial cells from the surface of the solution. Red blood cell lysis was performed if necessary, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were incubated with anti-CD3e+-Brilliant violet-421 , anti-TCRb+-APC antibodies, and Fixable viabily dye -eFlour 780. CD3e+ TCRb+ cells were isolated by FACS AriaIII. Library was performed according to the manufacter's instructions (Chromium Next GEM SIngle Cell ATAC Reagent Kits v2, 10x Genomics). The isolated nuclei suspension was diluted and then incubated with transposition mix for a targeted nuclei recovery of 10,000 cells. GEMs were then captured on the chromium NEXT GEM Chip H (10X Genomics 2000180). Following GEM incubation, clean up was performed using Dynabeads MyOne Silane beads (10X Genomics 2000048) and SPRIselect reagent (Beckman Coulter B23318). Finally, the library was amplified for a total of 7 Sample Index PCR cycles (WT, A3; KO, A4).