GSM8423558: Head_Dermis_05; Mus musculus; RNA-Seq
Skin was soaked overnight at 4℃ in a solution of 2000PU / mL DISPASEII (FUJIFILM Wako). After separating the epidermis from the dermis, the epidermis was washed with MEMα (Thermo Fisher Scientific, Waltham, MA) supplemented with 2% FBS and tissue was minced and incubated with 2-3 mL TryPLE Select (1X)(Thermo Fisher) supplemented with 1U DNaseI. After incubation in 37℃ water bath for 30-40 minutes, the cell supernatant was collected to the new tube through the 70 µm cell strainer. After centrifuging the tubes at 300g for 5-10 min, the pellet was obtained and suspended in 200 µL of MEMα supplemented with 2% FBS. RNA-seq library was constructed using a modified sciRNA-seq protocol. The target cells were sorted into 384-well plates with barcoded oligo dT primer for each well (1 cell / well). After reverse transcription in each well, cDNA was pre-amplified using the xGen™ ssDNA & Low-Input DNA Prep (IDT). Purified cDNAs were tagmentationed with Amplicon Tagment enzyme (Illumina) and PCR for library construction was done by using NEBNext High-Fidelity 2× PCR Master Mix (NEB).