title: GSM8703558: Orthotopic implanted tumor, Thbs1KO mouse,  rep2; Mus musculus; RNA-Seq
library_construction_protocol: Tumors generated by orthotopic implantation of MTO in the rectum of WT, Thbs1-/- and Thbs2-/- mice (n = 2 per group) were processed into single-cell suspensions using the Tumor Dissociation Kit (Miltenyi Biotec). Red blood cells were removed from the dissociated tumors using the Red Blood Cell Lysis Solution (Miltenyi Biotec), and dead cells were eliminated with the EasySep Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies). scRNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3' v3.1Kit (10X Genomics). Cells were loaded onto the 10X Chromium Controller (10X Genomics) with the Chromium Next GEM Chip G Single Cell kit (10X Genomics) at a concentration of 1,000 cells per µL, as described in the manufacturer's protocol (10X Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index)). A total of 16,500 cells from each mouse were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were performed according to the manufacturer's protocol. Individual sample quality was checked using a TapeStation (Agilent Technology). The final library pool was sequenced using the DNBSEQ-G400RS (MGI).