singlecell sequencing of CD45+ cell after PBS
Fresh tumors in the groups of PBS and mild PTT were harvested and stored in GEXCOPETM tissue preservation solution (Singleron, China) before dissection. Tumors of three mice were dissociated and pooled together to generate the single cell suspension. Firstly, specimens were washed with PBS and cut into 1-2 mm pieces. Then, the tissue pieces were digested with GEXSCOPETM tissue dissociation solution (Singleron, China) at 37 for 30min with agitation. Next, the mixtures were filtered through 70 m cell strainers for single cell suspension and washed with PBS. GEXSCOPETM red blood cell lysis buffer (Singleron, China) was added at room temperature and incubation for 15 min to remove red blood cells. The solution was then centrifuged at 500 g for 5 min and re-suspended in PBS followed by cell sorting with FACS Fusion sorter (BD bioscience, USA). Alive CD45+ immune cells were sorted and purity evaluated. The overall cell viability was confirmed by trypan blue exclusion, which needed to be above 85%. Single-cell suspensions (1105 cells/mL) with PBS were loaded into microfluidic devices using the Singleron Matrix Single Cell Processing System (Singleron, China). Then, the scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE Single Cell RNA Library Kits (Singleron, China)