title: GSM8965815: Langerhans cells extracted from mice ear skin treated with actone/croton oil/ olive oil; Mus musculus; RNA-Seq
library_construction_protocol: Mice were treated as described above, while control mice received only Nair treatment at the same time as experimental animals. The procedure was performed 48 hours after the initial treatment in both the croton oil and microneedle punch. Mice were then euthanized following standard protocols after anesthesia with isoflurane. Subsequently, ears were excised, and the skin layer was carefully separated from the underlying cartilage. The isolated skin was placed epidermis-side up in a trypsin solution (0.3% trypsin (Thermofisher; 15090046) in 150 mM NaCl, 0.5 mM KCl and 0.5 mM glucose) and incubated for 1 hour and 45 minutes at 37 °C. Following incubation, the epidermis was gently separated from the dermis using fine forceps. The epidermal sheet was transferred to a scratched-wall plastic Petri dish (Fisher; FB0875711YZ) containing HBSS buffer supplemented with 10% FBS, trypsin inhibitor (Sigma; T6522) and DNaseI (Sigma; D4513), pre-warmed to 37 °C. The epidermis was then mechanically dissociated by pressing it against the scratched surface using forceps, producing cloudy cell suspension. This suspension was incubated for 15 minutes at 37 °C, then filtered through a 70 µm cell strainer. Dead cells were removed via magnetic separation (Miltenyibiotec; 130-090-101), followed by magnetic enrichment of LCs (Miltenyibiotec; 130-095-408). Cell viability was assessed using either trypan blue staining or flow cytometry. For single-cell RNA sequencing, libraries were prepared according to the manufacturer's manual using Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, USA). And sequenced using 2 lanes (2.5GB) NovaSeq X 10B 100 SR (Illumina, USA).