GSM5457224: 2012364_Mouse-2 OR Mouse-3 Blood J35+70; Mus musculus; OTHER
Blood was drawn 7 days before and 34 days after PBC induction. One hundred five days after cholangitis induction (equivalent to 70 days after the engraftment of the CCA cells), blood, liver and the CCA tumor were collected from a PBC mouse. A soft red blood cell lysis protocol was used to preserved cell fitness and viability. Briefly, 500µL of PBS was added onto blood, followed by 3.5mL of sterile water. After mixing, 400µL of PBS 10x was included before centrifugation. Cells were resuspended in PBS containing 0.5% BSA and 5mM EDTA. Liver preparation. Perfused livers were cut into small pieces and transferred to GentleMACSTM C tubes (Miltenyi Biotech) containing 3mL of CO2 independent medium. After mechanical dissociation using GentleMACSTM Octo Dissociator (Miltenyi Biotech), the cell suspension was transferred into 50mL tubes for extensive washing. Finally, the cell pellet was resuspended in PBS containing 0.5% BSA and 5mM EDTA. Tumor preparation. Tumors were cut into small pieces and placed into GentleMACSTM C Tubes. Then, a cocktail of tissue-dissociating enzymes resuspended in 2.35mL of RPMI medium was added (Mouse tumor dissociation kit, Miltenyi Biotech). GentleMACSTM C Tubes containing the tissue were placed and incubated at 37°C onto a GentleMACSTM Octo Dissociator equipped with heaters (Miltenyi Biotech) for dissociation to occur. Next, cells were filtered through a 100µm cell strainer and washed twice in PBS. Finally, the cell pellet was resuspended in PBS containing 0,5% BSA and 5mM EDTA. T cell sorting. CD4+ and CD8+ T cells from blood, liver and tumor were sort-purified in a two-step procedure in which T cells were initially enriched in CD3+ cells using MoJoSortTM mouse negative selection kit (BioLegend). Then, CD3+ cells were stained with a cocktail of antibodies targeting CD11c, Ly6C, Ly6G, CD19, F4/80, NK1.1 (all in FITC for Lineage negative exclusion), CD45, CD8, and CD4, together with DAPI, then sorted using BD FACSAria III as DAPI-, Lineage-, CD45+, CD4+ or CD8+ cells. After sorting, cells were counted, then CD4+ and CD8+ cells were mixed at 1:1 ratio. Final concentration was adjusted to 800 cells/µL (400 CD4+:400 CD8+ cells/µL) scRNA-seq was performed on the 1:1 CD4+:CD8+ T cell mixture prepared from blood, tumor or liver. A total of 10000 cells per tissue were loaded onto the NextGEM 10x chip (10x Genomics). The RNA capture, barcoding, cDNA amplification and library were performed according the manufacturer's recommendations (CG00207_Chronium NextGEM SingleCellV(D)J v1.1 user guide). 10x libraries were sequenced on NovaSeq (Illumina) SP (for TCR-seq) or S1 (for gene expression).