GSM3668833: e12MYC-108; Mus musculus; RNA-Seq
Tissue was treated overnight at 4C in dispase. Epidermis was separated from dermis and dermis was discarded. Epidermis was then treated 20 min at 37C in 1:1 mix of trypsin/versene. Tissue was then homogenized and filtered to obtain a single cell suspension using 70um cell strainers. Single cell suspensions were stained with AnnexinV (Alexa Fluor 568 conjugated, Thermofisher, 1:200) and sorted into 96-well plates using a BD FACSAriaII. CD45 (biotinylated rat anti CD45, BD Biolegend, 1:200), CD117 (biotinylated rat anti CD117/c-kit, Biolegend, 1:200), CD31 (biotinylated rat anti-CD31/PECAM, Bioscience, 1:200), and CD140a (biotinylated rat anti-CD140a, Biolegend, 1:200) were used as lineage negative markers (to exclude immune cells, melanoblasts, endothelium, and fibroblasts, respectively). All lin- were detected with a strepdavidin-conjugated FITC or APCCy7 secondary (Biolegend, 1:500). Single cell RNA-seq libraries were prepared using the Smart-seq2 protocol (see reference 40) with a few modifications as follows: plates were thawed on ice and incubated for 3 min at 72C to lyse cells. Reverse transcription (4U/ml Maxima H- transcriptase), template switching reaction and PCR pre-amplification (15 cycles) were performed according to the manufacturers' protocol. PCR products were cleaned up using 0.8X AMPure XP beads (Beckman Coulter). The cDNA library quality was assessed by qPCR for primer pairs targeting the housekeeping gene, Gapdh. Samples showing high Ct value (>35 cycles) or having no PCR amplification were excluded from downstream analysis. 50-100pg of each cDNA library was used for generating Illumina sequencing libraries using a Nextera XT DNA library preparation kit (Illumina). After the final PCR amplification step, samples were pooled and cleaned by 0.9X AMPure XP beads. The pooled sequencing libraries were sequenced in the RU genomics facility on an Illumina Nextseq500 instrument using the High Output 75 PE kit.