title: GSM8626612: C57BL/6N_mouse_skin_blood_bone_marrow_14_days_after_Leishmania_mexicana_infection; Mus musculus; RNA-Seq
library_construction_protocol: Foot skin: Whole foot skin was cut into small pieces which were digested for 45-60 minutes at 37°C under gentle vibration in RPMI 1640 containing 0.2 mg/ml collagenase P and 0.1mg/ml DNAse I (both Roche Diagnostics GmbH). Cell suspensions were passed through a 70 µm and 40 µm cell strainer (Corning), resuspended in 2.5 ml of 40% Percoll preparation (20 ml Percoll [GE Healthcare], 2.2 ml 10×PBS, 27.8 ml HBSS without Ca2+ and Mg2+ [Sigma-Aldrich]) and layered onto 1.5 ml 60% Percoll (30 ml Percoll, 3.3 ml 10×PBS, 16.7 ml HBSS). After centrifugation for 20 min at 931 x g (room temperature), the cell layer at the interface was collected, washed and resuspended in PBS. Viability was increased by removing dead cells using the MACS Dead cell removal kit (Miltenyi Biotec) according to the manufacturer's instructions. Bone marrow (BM): Femur and tibia were flushed using complete RPMI medium and a 23-gauge needle (BD Biosciences). The content was collected, filtered through a 70 µm cell strainer and red blood cells were lysed using Red Blood Cell Lysis Buffer (Miltenyi Biotec) according to the manufacturer's protocol. A density gradient centrifugation was then conducted using Histopaque 1077/1119 (Sigma-Aldrich), following the manufacturers protocol. Afterwards, the granulocyte layer was carefully transferred and washed with PBS. The viability was increased by removing dead cells using the MACS Dead cell removal kit according to the manufacturer's instructions. Blood: Following euthanasia of the mice, a midline laparotomy was performed to expose the abdominal cavity, and the vena cava was carefully isolated and accessed using a 26G needle attached to an Omnfix-F Luer Solo syringe (Braun). Blood was drawn from the vena cava and transferred into a  EDTA-coated tubes (Micro tube 1.3 mL, Sarstedt).A density gradient centrifugation was then conducted using Histopaque 1077/1119 (Sigma-Aldrich), following the manufacturers protocol. Afterwards, the granulocyte layer was carefully transferred and washed with PBS. Red blood cells were lysed twice in ice-cold water for 10 seconds. The viability was increased by removing dead cells using the MACS Dead cell removal kit according to the manufacturer's instructions. Discrimination between viable and dead cells was enabled by 5 μM Calcein AM and 0.3 μM DRAQ 7, as described Single-Cell Capture and cDNA Synthesis Protocol for BD Rhapsody (BD Bioscience). Samples which were subsequently pooled were labelled with sample tags (BD Bioscience, BD Mouse Single-Cell Multiplexing Set, 626545, Rat anti-mouse MHC-H2 Class I(M1/42)) according to manufacturer's protocol. Cell loading in the BD Rhapsody 8-Lane Cartridge (BD Bioscience) and single-cell capture with the BD Rhapsody HT Single-Cell Analysis System was performed as mentioned in the manufacturer's instructions. Single-cell whole transcriptome mRNA and Sample Tag libraries were generated according to the manufacturer's instructions (BD Rhapsody, BD Bioscience). The final libraries were quantified using a Qubit Fluorometer (Thermo Fisher Scientific) with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).