Single cell analysis of CD45 positive cells isolated from MC38 colon carcinoma tumor-bearing C57BL/6 mice
Single cell analysis of CD45 positive cells isolated from MC38 colon carcinoma tumor-bearing C57BL/6 mice
Tumor cells were separated from the C57BL/6 mice on the 10th of tumor growth, Tumor Dissociation Kit (Miltenyi,130-096-730) was used to dissociate the tumor tissues into single cells as the manufacturer's instructions. ACK lysis buffer (Gibco,492-01) was used to remove the red blood cell. Then Dead Cell Removal kit (Miltenyi,130-090-101) was used to remove dead cells by column in MACS Separator, and CD45+ cells were enriched from tumor tissues according to CD45(TIL) MicroBeads (Miltenyi,130-110-618).The cells were resuspended with 1×PBS containing 0.04% BSA, and cell viability was detected with 0.4% trypan blue staining solution and counted with Countess II (Invitrogen) cell counter, and the cell viability was over 90%. The cell suspension was loaded into Chromium microfluidic chips with 30v3 chemistry and barcoded with a 10× Chromium Controller (10X Genomics). mRNA from the barcoded cells was reverse-transcribed into cDNA according to the manufacturer's instructions . Sequencing library was constructed according to the instructions of Chromium Single Cell 3ʹ Reagent kits v3 (10×Genomics).