title: GSM9499534: scRNA_Spleen; Mus musculus; OTHER
library_construction_protocol: 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. Fresh tumor samples were digested in an enzyme mixture (DMEMc (Dulbecco´s modified Eagle´s medium (Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (PAN-Biotech, Aidenbach, Germany) and 100 U/mL penicillin‒streptomycin (Thermo Fisher Scientific)) supplemented with 0.2 μg/mL dispase, 0.2 μg/mL collagenase A, and 100 μg/mL DNase I (Merck, Darmstadt, Germany)), with 1 mL per sample for 45 min at 500 rpm and 37 °C. The cells were passed through 100 μm sterile strainers (Cell Trics, Partec, Sysmex Europe GmbH, Goerlitz, Germany) and centrifuged at 300 × g and 4 °C for 5 min, after which the supernatant was discarded. Bone marrow, splenic or tumor cells 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. After extensive washing, cells were 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. he HTO counts are appended to the end matrix.mtx.gz files, with identifiers in the features file starting with "HTO...".