title: GSM9621658: B6 HP tumor organoid cells; Mus musculus; RNA-Seq
library_construction_protocol: B6-HP tumor organoids were s dissociated using TrypLE. Cell viability was assessed using Trypan blue staining. Cells were then resuspended in PBS containing 0.04% BSA, with a viability of 82%. The cell suspension was centrifuged at 400 × g for 5 minutes at 4 °C and resuspended to a final concentration of 4,900 cells/µL in a total volume of approximately 90 µL. Utilizing the Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead kit v.3.3 and Chromium instrument, cells were partitioned into nanoliter droplets to achieve single-cell resolution for a maximum of 10,000–15,000 individual cells per sample (10x Genomics). The resulting cDNA was tagged with a common 16-nt cell barcode and 10-nt unique molecular identifier (UMI) during the reverse transcriptase (RT) reaction. Full-length cDNA from poly-A mRNA transcripts was enzymatically fragmented and size selected to optimize the cDNA amplicon size for library construction (10x Genomics). The concentration of the 10x single-cell library was accurately determined through qPCR to produce cluster counts appropriate for the NovaSeq-6000 platform (Illumina). The sequence data were generated targeting 50,000 read pairs per cell, which provided digital gene expression profiles for each individual cell.