title: GSM9342348: GEX library (multiplexed SHAM, IMQ, IMQ + GSKJ-4), batch1; Mus musculus; RNA-Seq
library_construction_protocol: Spleen was disrupted onto a filter mesh in a 3 cm Petri dish. Red blood cells were lysed using commercial RBC lysis buffer by incubation at room temperature for 3 minutes, followed by centrifugation for 2 minutes at 4°C and 1000 rcf. Dorsal skin was excised, manually cut into small pieces using scissors and digested in DMEM, 4 mg/ml collagenase type IV, 20 μg/ml DNase I and 5 mg/ml bovine serum albumin. Skin tissue was digested using the gentleMACS C tubes and the program '37C_Multi_H'' on the gentleMACS dissociator was performed. Murine samples were stained using surface anti-mouse antibodies. Individual samples were stained with 6 TotalSeq™-C anti-mouse hashtag antibodies at 4°C for 20 minutes in 100 μL FACS buffer. CD45⁺ immune cells were sorted for single-cell RNA sequencing. Single-cell libraries were prepared according to the manufacturer's protocol (Chromium Next GEM Single Cell 5′ v2 with Feature Barcode Technology, CG000330 Rev F). Following sorting, cells were centrifuged at 300 × g for 5 minutes at 4°C, and the supernatant was carefully removed. The resulting cell pellets were resuspended in 37.5 µL of 0.5% BSA-PBS buffer. Cell suspensions were mixed with Master Mix from the Chromium Next GEM Single Cell 5′ v2 (Dual Index) kit with Feature Barcode Technology (10x Genomics), and samples were loaded onto a Chromium Next GEM Chip K (10x Genomics).