A5, replicate1, scRNAseq
Three days after the last 3A5 BiTE injection, spleen samples were collected from the mice and dissociated by TissueMaster High-Throughput Tissue Homogenizer. Single cells were obtained through a 70 m cell strainer, and the cells were washed with RPMI-1640/2% FBS, followed by PBS. Excess erythrocytes were removed with the red blood cell lysis buffer (Solarbio, Beijing, PR China, cat# R1010), then the remaining cells were resuspended in RPMI-1640/2% FBS, and their viability was detected with trypan. After the removal of dead cells using the Dead Cell Removal Kit (Miltenyi Biotec, Germany, cat#130-090-101), CD45+ immune cells were sorted from single-cell suspensions using CD45 (TIL) Microbeads (Miltenyi Biotec, Germany, cat#130-110-618), according to the manufacturer's protocol. Cells were used for scRNA-Seq library construction when more than 85% of cells were in good condition, as determined by bench blue staining. The single-cell sequencing was performed using Chromium Next GEM Single Cell 3 Reagent Kits v3.1 (10x Genomics) following the manufacturer's protocol.