scRNA-seq of circulating tumor cells-derived xenograft
To generate CDX models, PDX-CTCs were needle-injected (s.c. in flanks) with 30% Matrigel in 200 L of CTC media into nave NSG mice. Snap-frozen matched PDX and CDX tumor tissues were minced on ice and homogenized using Tissue-Tearor (Biospec) in cold Nuclei pure Lysis Buffer (Millipore-Sigma) containing 0.1% Triton X-100 and 0.2 U/L RNase inhibitor (Promega). Homogenate was transferred through 70-m cell strainer, homogenized again a few strokes, and passed through 40-m cell strainer. Filtrate was centrifuged at 500 g for 5 min at 40 C and re-suspended the pellet in PBS with 0.4% BSA and 0.2 U/L RNase inhibitor. This step was repeated twice to wash the nuclei and finally counted after staining with Trypan blue using Countess II FL Automated Cell Counter (ThermoFisher Scientific). Single-nuclei 3 RNA-seq (snRNA-seq) library preparation and sequencing was provided by the University of Missouri DNA Core Facility. Extracted nuclei suspended in 0.4% BSA in PBS with 0.2 U/L RNase inhibitor at a concentration of 800 nuclei/L was used for a targeted capture recovery of 3,000 nuclei per sample using the 10x Genomics Chromium System. Following encapsulation in a lipid droplet, cDNA and library generation was performed according to the manufacturers protocol using the Chromium Single Cell 3 Library Gel Bead kit (v2). Sequencing was performed using an Illumina NovaSeq 6000 to an average of 50,000 reads per nuclei.