GSM7230210
At day 20 post-implantation tumors were dissociated using Miltenyi tumor dissociation kit (mouse) (#130-096-730) and the gentleMACS Octo Dissociator with heaters from Miltneyi Biotec.Briefly, Tough tumor dissociation protocol was used, program 37_m_TDK_2 followed by program  m_imptumor_01 12,000 cells of the resulting cell suspensions were submitted for single-cell RNA-seq library preparation at the Cancer Research UK Cambridge Institute Genomics Core Facility. Chromium Single Cell 3′ Library & Gel Bead Kit v3, Chromium Chip B Kit and Chromium Single Cell 3' Reagent Kits v3 User Guide (Manual Part CG000183 Rev A; 10X Genomics). Cell suspensions were loaded on the Chromium instrument with the expectation of collecting gel-beads emulsions containing single cells. RNA from the barcoded cells for each sample was subsequently reverse-transcribed in a C1000 Touch Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to the manufacturer's protocol with no modifications (12 cycles was used for cDNA amplification). cDNA quality and quantity was measured with Agilent TapeStation 4200 (High Sensitivity 5000 ScreenTape), after which 25% of material was used for gene expression library preparation. Library quality was confirmed with Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library sizes) and Qubit 4.0 Flourometer (Thermo Fisher Qubit™ dsDNA HS Assay Kit to evaluate dsDNA quantity). Each sample was normalized and pooled in equal molar concentration. To confirm concentration pools were qPCRed using KAPA Library Quantification Kit on QuantStudio 6 Flex before sequencing.