GSM4475240: RNA young rep1; Mus musculus; RNA-Seq
Skin pieces were incubated in 0.8% trypsin in PBS for 60 minutes at 37°C. After incubation, the epidermis was manually detached from the dermis, single cell epidermal suspensions where generated by pippeting and collected by centrifugation. Cells were labeled with antibodies against Cd34 and alpha6 integrin, after which Cd34 alpha6 integrin-double positive cells were isolated by flow cytometry. Total RNA was isolated using NucleoSpin RNA Plus kit for RNA seq. For ATAC seq Briefly, nuclei of 50K freshly FACS-sorted single HFSCs were prepared by lysing cells with cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0,1% IGEPAL CA-630) for 15 min on ice. Nuclei were then collected by spinning at 6000 g for 10mins at 4°C, resuspended in Tagment DNA Buffer and Tagment DNA Enzyme (TruSight One Kit) and incubated for 30 min at 37°C. Samples were purified with MinElute PCR purification kit (QIAGEN) and PCR-amplified with 10-12 cycles. Tagment DNA TDE1 Enzyme and Buffer Kits (Illumina) for ATAC seq; Ovation RNA-Seq System V2 (NuGen) followed by subsequent library preparation using the Nextera XT library preparation kit (Illumina) for RNA seq