GSM8594730: Skin, depleted, rep2; Mus musculus; RNA-Seq
The excised skin from MAFIA mice was cut into small pieces and incubated in 5 mL of dissociation buffer (5 mL RPMI 1640 (no calcium or magnesium) with 232U DNAse I, 0.25 mg/mL Liberase stock (2.5 mg/mL), 23.2 mM HEPES, and 2.32mM sodium pyruvate) at 37°C for 60 minutes. The digestion was stopped by adding 10 µL of 0.5 M EDTA and 400 µL FBS, and the samples were transferred to GentleMacs tubes (Miltenyi Biotec, Cat#130-093-237) for dissociation (program: m_impTumor_01.01). The digested samples were filtered through a 70 µm filter, resuspended in 4 mL of FACS buffer (500 mL PBS, 2 mL of 0.5 M EDTA, 25 mL FBS, and 5 mL non-essential amino acids (100x stock)) and stained with viability dye (Propidium iodide). Live cells were sorted by DiVa cell sorter (BD Biosciences). 18K sorted PI- live cells were loaded for scRNA-seq library preparation. The live cells were sorted and loaded on the 10x Genomics Chromium using the Chromium Single Cell 3' Reagent V3 Kit and the sequencing libraries were constructed following the user guide.