Fresh tissue samples were promptly submerged in DMEM with 10% FBS at 2-8 °C upon collection. Prior to tissue digestion, the samples were rinsed with Hanks Balanced Salt Solution (HBSS) and sliced into 1-2 mm fragments. Subsequently, the tissue fragments were incubated in 1mg/ml collagenase IV (Thermofisher # 17104019) for 30 minutes and neutralized with HBSS containing 2% FBS. Following filtration through a 70µm cell strainer, the cells were centrifuged and prepared for sequencing without prior sorting. Concurrently, to monitor the decontamination process of our tissue samples, the HT29 cell line (ATCC, # HTB-38™) was employed as a control throughout the sequencing procedure. Cell counts and viabilities were determined using trypan blue exclusion and counted on a Countess 3 FL automated cell counter (Life Technologies). Cell capture processing and gene expression library preparations were performed following 10X Genomics' guidelines for 5' scRNA-seq. QC steps after cDNA amplification and library preparation steps were carried out by running ThermoFisher Qubit HS dsDNA Assay along with Agilent (Santa Clara, CA) HS DNA Bioanalyzer for concentration and quality assessments, respectively. Equal library amounts were used for pooling. Library pool concentration was verified using qPCR using a KAPA Biosystems KAPA Library Quantification Kit prior to sequencing. The pool was sequenced using three lanes of a NovaSeq6000 S4-XP, 200-cycle flow cell. The run parameters used were 26 cycles for read 1, 90 cycles for read2, 10 cycles for index1, and 10 cycles for index2 as stipulated in the protocol mentioned above. Sequencing depth targeted was 100,000 reads/cell. cDNA