GSM9535550: Pparg-/-epi; Mus musculus; RNA-Seq
Two mice (one male and one female) were euthanized for each genotype (wildtype and Pparg-/-epi). Dermal and epidermal cells were isolated from areas of telogen phase hair cycling for each mouse. For dermal cell preparation, the tissues were minced and digested in 2.5 mg Liberase TM, 16.7 mg DNase I, penicillin, and streptomycin in RPMI media (all reagents from Sigma-Aldrich, St. Louis, MO, USA) for 90 min at 37 °C. The digest was filtered through a 40 μm filter. After adding 5 mL of ice-cold dialyzed FBS, the samples were centrifuged at 300× g for 10 min. RBC lysis (RBC Lysis Buffer, Cat #00-4333-57, ThermoFisher Scientific, Waltham, MA, USA) followed by dead cell removal (dead cell removal kit #130-090-101, Miltenyi Biotec, Auburn, CA, USA) with centrifugation were performed after each step. The cell prep was washed twice with cell suspension buffer [PBS (calcium, magnesium, DNase, and RNase free) (Catolog # 40120706, bioWORLD, Dublin, OH, USA) containing 1% BSA] followed by centrifugation. RNA sequencing was then performed on a final cell prep resuspended in cell suspension buffer containing 0.5 U/µL SUPERaseIn™ RNase Inhibitor (Catalog #AM2694, ThermoFisher Scientific, Waltham, MA, USA) and 0.5 U/µL Protector RNase Inhibitor (Catalog #3335399001, Sigma-Aldrich, St. Louis, MO, USA). For epidermal cell preps, the dermal fat and dermis were removed using a scalpel, and the epidermal sheet was suspended in dispase II solution Sigma-Aldrich, St. Louis, MO, USA) for 2 hrs at 37 °C. After suctioning the dispase and scraping the epidermis from the dermis, the tissue was rinsed with PBS, centrifuged, and incubated in 5 mL Accutase solution (Cat# CnT-Accutase-100, CELLnTEC Advanced Cell Systems, Bern, Switzerland) for 20 min at room temp. After filtering through a 70 µm filter, 5 mL of dialyzed FBS was added, and the cells were pelleted by centrifugation. Dead cell removal and further processing were then performed as described for the dermal cell prep. The epidermal cells and dermal cells were then recombined at a 1:10 ratio (dermal vs. epidermal), and the male and female cells were pooled together. single cell RNA-seq libraries were prepared using the 10X Genomics Chromium NextGEM Single Cell 3' V3.1 kit.