NextSeq 500 paired end sequencing; Single cell RNA sequencing of mouse macrophage
Single cell RNA sequencing of mouse macrophage
For extraction of mouse peritoneal macrophage, we followed a previously described protocol. A total of 5 mice (3 from BALB/c and 2 from C57BL/6) were used for the study. In short, the mice were euthanized using CO2 as per the protocol validated by the ethics committee. The abdomen of the mouse was cleaned with 70% ethanol. A small incision was made at the outer skin of the peritoneum and the inner skin lining of the peritoneum was exposed. Five ml of ice-cold 3% fetal calf serum (FCS) in 1X PBS was injected into the cavity using a 27G needle. The peritoneum was gently massaged to dislodge the immune cells in the cavity. A 25G needle was used to collect the fluid and was placed in a sterile ice-cold tube on ice. The peritoneal lavage was centrifuged at 200g for 5 min, and resuspended in RPMI-1640 medium supplemented with 10% heat inactivated FCS. The cells were plated in a 10 cm plate at 37 degrees Celsius and 5% CO2. The plates were washed 3 times after 6 hours with 1X PBS supplemented with 3% FCS to remove the floating cells. The adherent cells were scraped and used for 10X Genomics Single cell transcriptomics library preparation. The abdomen of the mouse was cleaned with 70% ethanol. A small incision was made at the outer skin of the peritoneum and the inner skin lining of the peritoneum was exposed. 10X Genomics Single cell transcriptomics library preparation according to the manufacturer's protocol. 10X Genomics Single cell transcriptomics library preparation according to the manufacturer's protocol.