GSM8283993: AL7361-T1, TCR; Mus musculus; OTHER
Mice were sacrificed and then the liver was perfused with PBS+EDTA 2mM through the inferior cava vein. Individual tumor nodules were visually inspected and selected to avoid healthy adjacent tissue and normalize for both size and level of expected necrosis across all samples. The selected tumor was then extracted from the liver using a sterile scalpel, weighed, and manually minced with sterile instruments. Minced tumor nodules were then enzymatically digested with 2.5mL enzymatic digestion mix (Miltenyi Biotech,Tumor dissociation Kit) in DMEM medium in a gentleMACS C Tube (Miltenyi Biotech) and then placed on gentleMACS Octo Dissociator (Miltenyi Biotech) for enzymatic and further mechanical dissociation with the “37°C_m_TDK1” program. Lysates were strained through a 70-µm cell strainer (Miltenyi) and resultant single cells were pelleted at 300g for 5 min at 4°C. From this point onward, cells were kept at 4°C. Elimination of red blood cells using “Red blood cell lysis solution” (Miltenyi Biotech) for 2 min on ice followed by elimination of dead cells using the “Dead Cell Removal” kit (Miltenyi Biotech) was performed according to manufacturer protocols. CD45 positive cells (immune cells) were separated from CD45 negative cells (tumor and non-immune stromal cells) using the CD45 microbeads mouse kit (Miltenyi Biotech) and each fractions were counted manually using a hemacytometer, then recombined at 1:1 ratio. This step was performed to ensure sufficient numbers of immune cells were sequenced from the bulk tumor samples. Only samples with a viability >80% were used. Single-cell RNA sequencing (scRNA-seq) sample processing was immediately performed after cell extraction/isolation by the HIMC core facility at the Icahn School of Medicine at Mount Sinai. Two single-cell suspensions were counted (Nexcelom Cellometer Auto 2000) and each loaded on one lane of the 10x Genomics NextGem 5'v2.0 assay as per the manufacturer's protocol with a targeted cell recovery of 10,000 cells per lane. Gene expression (5'Gex) and TCR libraries were generated as per the 10x Genomics demonstrated protocol (https://assets.ctfassets.net/an68im79xiti/57JaTECQNBPSpyDz8 oucdi/ced6aa8eaf73d6ee18dea8fdbd945faa/CG000331_Chromium_Next_GEM_Single_Cell_5-v2_UserGuide_RevD.pdf). All libraries were quantified via Agilent Technologies 4200 Tapestation and KAPA library quantification kit (Roche). Gene expression libraries were sequenced at a targeted depth of 25,000 reads per cell and TCR libraries at a targeted read depth of 5,000 reads per cell. Libraries were sequenced on the Illumina NovaSeq S2 100 cycle kit with run parameters set to 26x10x10x90 (R1xi7xi5xR2).