GSM7107648: CCR2+ macrophages, hypodermis, replicate 2; Mus musculus; RNA-Seq
Back skins from C57BL/6 female adult mice were harvested after shaving with an electric clipper. Subcutaneous fat was removed from hypodermis and hypodermal layers were physically isolated from the dermis with forceps. Dermis was isolated from epidermis after 45min of an enzymatic treatment with 0.15% Trypsin/0.75mM EDTA. Isolated dermis and hypodermis were each minced with scissors and digested for 2H at 37°C in RPMI supplemented with 5% FCS and containing 0.25mg/mL of liberase T-Flex and 1ug/mL of DNase I. Cells were isolated from each layer and stained with antibodies as well as with propidium iodide (PI) before being FACS sorted as dendritic cells (PI–, singlet, CD45+, CD11b+, Ly6c-lo, MHCII-hi, CD64–), CCR2+ macrophages (PI–, singlet, CD45+, CD11b+, Ly6c-lo, CD11c-mid/lo, CD64+, CCR2+) and CCR2– macrophages (PI–, singlet, CD45+, CD11b+, Ly6c-lo, CD11c-mid/lo, CD64+, CCR2–). Cells were then washed, counted and used for RNA-sequencing. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. Libraries were prepared using the SMART-Seq Ultra Low Input RNA kit with Nextera XT library prep protocol according to the manufacturer's instructions (Illumina, San Diego, CA, USA).