GSM8787514: CAC227_S3_L001; Homo sapiens; RNA-Seq
To isolate nuclei from the in vivo co-cultures we utilized the protocols created by Habib et al. 21 Briefly, sorted dissociated xenograft cells were washed and suspended in ice cold nuclei EZ lysis buffer (Sigma, Cat#NUC-101) and homogenized using a glass dounce tissue grinder (20 times per passage for both a loose and tight pestle) and incubated on ice for 5 minutes following the addition of 2 mL of ice-cold nuclei EZ lysis buffer. The nuclei were centrifuged at 500g for 6 minutes at 4 Celsius, washed with nuclei EZ lysis buffer and filtered through a 40 µm strainer. The nuclei are then resuspended in 4 mL of nuclei suspension buffer (1x PBS, 0.01% BSA and 0.1% RNAse inhibitor) and filtered through a 20 µm strainer. Nuclei were counted and then processed according to 10x Genomics Single Cell 3' v3 kits in preparation for sequencing. library was constructed using the 10x Genomics Single Cell 3' v3 kits.