GSM8001709: Red pulp macrophages, naïve, WT; Mus musculus; RNA-Seq
Mice were humanely euthanized utilizing CO2 displacement and cervical dislocation. Bone marrow-derived monocytes were recovered by flushing the bone marrow of femur plus tibia with ice-cold FACS buffer (PBS-/- supplemented with 2% heat-inactivated FCS and 2mM EDTA). To obtain splenocyte single-cell suspensions, spleens were crushed through a 70 µm filter using a syringe and suspended in 4 ml ice-cold FACS buffer. For peritoneal lavage, 5-7 ml ice-cold FACS buffer was injected into the peritoneal cavity followed by abdominal massage. Buffer containing peritoneal cells was aspirated by syringe equipped with a 20G needle. All cell suspensions were subjected to red blood cell (RBC) lysis using a commercially available buffer (Sigma). Cells were pelleted by centrifugation (300 x g, 5 min, 4˚C), resuspended in ice-cold FACS buffer, and subjected to cell count determinations. Flow cytometry-based isolation of Lin-CD11b+CD115+Ly6G-Ly6C+ bone marrow (BM)-derived monocytes; Lin-F4/80+CD11bintMHCIIhi red pulp macrophages from digested spleens; Lin-F4/80+CD11b+Ly6Clo peritoneal macrophages; MerTK+CD64+ B16 tumor-associated macrophages; all cell populations were derived from WT and Pirb-/- (PirB KO) female littermate mice and immedeiately processed and subjected to scRNA-seq. Single-cell suspensions were prepared according to the Chromium Single Cell 3' Reagent Kits User Guide (v2 Chemistry). Samples were loaded onto a 10x Chromium instrument to produce sequencing libraries, which were processed according to methods provided by 10x Genomics. Cells were sequenced and processed to generate expression matrices using Cell Ranger (10x Genomics). Libraries were prepared using the 10X Genomics Chromium Single Cell 3' v4 Chemistry Reagent Kit, as per the parameters recommended by 10X Genomics.