title: GSM9460834: Stromal cells, K/BxN serum transfer day 5; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized and synovial macrophages were isolated from the hind paws. After removal of skin, muscles and tendons, the paws were digested using 2.5 ml RPMI-1640 (Roswell Park Memorial Institute) medium (Life Technologies), 10% heat-inactivated fetal calf serum (FCS, Life Technologies), 2 mg/ml collagenase IV from Clostridium histolyticum (Sigma-Aldrich, C5138), and 0.03 mg/ml desoxyribonuclease I (Sigma-Aldrich, DN25). Tissues were incubated for 30 min in an orbital shaker at 37°C with 300 rpm shaking. Afterwards the solution was filtered through a 40 µm mesh and digestion was stopped using 10 ml ice-cold MACS Buffer containing phosphate buffered saline (PBS, Life Technologies), 2% FCS and 2 mM EDTA (AccuGENETM, Lonza, 12001-382). The cells were then centrifuged for 7 min at 1600 rpm, 4°C. For cell sorting the pellet was resuspended in 3 ml of 40% Percoll (Sigma-Aldrich) solution and centrifuged for 15 min at 2200 rpm, room temperature, without acceleration and brakes. The pellet was washed once in MACS buffer and afterwards incubated for 15 min at 4°C with Fc block (Biolegend) followed by the extracellular antibody staining solution (final dilution of 1:400) for 20 min at 4°C with a washing step in between. Lastly, cells were resuspended in MACS buffer without EDTA for fluorescence-activated cell sorting. Sorted CD45+CD11b+Ly6G- or CD45- synovial cells from hind paws were used for single-cell sequencing. Cells for single-cell RNA sequencing (scRNAseq) were processed according to the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide (10x Genomics, CG000204) using the recommended Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1, (PN-1000128). The library was sequenced by Novogene with a depth of 20,000 reads per cell. Reads were aligned to the pre-built 10X mouse reference dataset (refdata-gex-mm10-2020-A)