GSM9456778: Enriched BCA, ZT1, 5 mice, GEX; Mus musculus; RNA-Seq
Mice were euthanized by ketamine/xylazine overdose, the blood was collected by heart puncture after which the mice were flushed with 10 ml of ice-cold PBS. Subsequently, organs were harvested and directly processed for functional assays or flow cytometry analysis. Blood was collected in a tube containing EDTA and incubated with erythrocyte lysis buffer (Biolegend) for 6 minutes on ice for erythrocyte lysis. After washing, cells were centrifugated and resuspended in HANKs buffer (HBSS w/o Mg and Ca + 0.06% BSA + 0.3mM EDTA). For isolation of bone marrow cells, the femur was harvested and flushed using 5 mL of HANKs buffer using a syringe with a 21G needle. Splenic cells were obtained by the mince and smashing of the spleen over a 70um filter.  For lung perfusion, heart and lungs from PBS-perfused mouse were gently harvested and placed on ice in a Petri dish. A needle was inserted into the right ventricle, at the level of the pulmonary artery, and lungs was slowly perfused with 20 ml of ice-cold PBS + 5 mM EDTA to detach adherent cells. For liver perfusion, PBS-perfused mice were placed on a cooling pad, the portal vein was canulated using a catheter and fixed in place with tissue adhesive (Surgibond). Once the catheter was fixed in place, the liver was gently harvested and placed on ice in a Petri dish. Liver was then slowly perfused with 20 ml of ice-cold PBS + 5 mM EDTA using a perfusion pump (2ml/min) to detach adherent cells. For isolating aortic cells, the brachiocephalic artery was isoalted, cleaned from surrounding fat and digested in 1 ml RPMI with 10% FCS and 1.25 mg/ml Liberase at 37 °C for 1 hour. Brachiocephalic artery (BCA), bone marrow, splenic cells as well as lung and liver perfusates were subjected to erythrocyte lysis in 1mL of erythrocyte lysis buffer (Biolegend) for 1 minute on ice. After washing, cells were centrifugated and resuspended in HANKs buffer. Samples were then normalized across conditions to obtain similar number of target cells (myeloid cell populations and progenitors). Normalized samples were then incubated with hashtag antibodies specific for each organ, mouse and condition (samples X1, X2, X4, X5) or only organ (samples X3, X6). After extensive washing, cells were pooled FACS-sorted. Sorted cells were then loaded into the 10x Genomic platform and processed with 10X Chromium Next GEM Single-Cell 3′ Reagent Kit with Feature Barcoding technology following manufacturer's instructions.