GSM6827323: Control, replicate 3, tumor, ADT-seq and CITE-seq; Mus musculus; OTHER
After treatment, tumours were dissociated into single-cell suspensions using a gentleMACS dissociator and enzymatically digested in a buffer containing collagenase D (2 mg/ml) and DNAse (40 U/ml).  Single-cell suspensions of draining lymph nodes were obtained by mechanical dissociation through 40 µm cell strainers and performing red blood cell lysis as needed.  Blood was obtained by terminal cardiac puncture and collected in lavender Microtainer Blood Collection Tubes and subjected to red blood cell lysis.  Blood samples were multiplexed using 10 barcodes with a distinct pair used to identify each individual sample before pooling. Tumor and blood samples were sorted by FACS for CD90+ T cells, and draining lymph node samples were sorted for CD90+CD44+ T cells. 5' Chromium Single Cell 5' Library and Gel Bead Kit (10X Genomics).  T cells were allowed to bind to tetramers of gp70 antigen bound to associated ADT barcodes.  CITE-seq was performed using antibodies against 18 selected proteins plus 6 control immunoglobulin proteins.  A total of 14 cycles of PCR amplification were performed.