GSM4629055: 1J_young_NMR_epidermis; Heterocephalus glaber; RNA-Seq
Epidermal cells from 3 young and 3 aged NMR were collected with punch biopsy devices on the backs of the animals. To preserve cell viability, skin pieces were immediately placed in PBS/BSA 3% medium on ice until dissection. Using a scalpel, the subcutaneous fat was removed from the dermis. The cleaned tissues were placed dermis side down in a sterile petri dish in a trypsin 0.25%/EDTA 1mM solution at 37°C for 1h. The enzymatic digestion was stopped by addition of PBS/FBS 10%. Epidermis was then mechanically dissociated and minced in small pieces with razor blades in PBS and then triturated. 3 rounds of washes were performed starting with 100µm filtration, 70µm and 40µm and interspersed with centrifugation cycles at 150g at 4°C for 5 minutes. Finally, the cell pellet was resuspended in PBS/BSA 3%. Single cells were captured in droplet emulsion using the Chromium Controller (10× Genomics, Pleasanton, CA), and scRNA-seq libraries were constructed according to the 10× Genomics protocol using the Chromium Single-Cell 3′ Gel Bead and Library V2 Kit (10× Genomics, Pleasanton, CA). All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with a 96 Deep Well Reaction Module. Twelve cycles were used for cDNA amplification and sample index PCR. Amplified cDNA and final libraries were evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip. Samples were sequenced on an HiSeq 4000 instrument (Illumina, San Diego, CA). ScRNA-seq
GEO Accession
GSM4629055