GSM4156451: Postnatal day 1 melanocytes; Mus musculus; RNA-Seq
Isolation of melanoblasts and melanocytes: FVB/N iDct-GFP dams were fed doxycycline-fortified chow for the entire duration of gestation until collection of E15.5, E17.5 and P1 pups. Doxycycline was injected intraperitoneally at 80 μg/g body weight 24 hr before collection of P7 pups. A single cell suspension was generated from embryos and skin of newborn pups. Multiple litters were used for each developmental stage, and embryos/pups from each stage were pooled to ensure adequate numbers of GFP+ cells. The head was removed to prevent collection of GFP positive cells in the embryonic telencephalon, and melanocytes from the inner ear or from the retinal pigmented epithelium (RPE) were discarded. Excess tissue was also removed. The spinal cord was kept intact as some melanoblasts still remain in the neural crest area. At E17.5, P1 and P7 stages, most melanocytes have reached the dermis, thus only the skin was collected from these developmental stages. Back skin was immersed in a shallow layer of 1X PBS and subcutaneous fat was scraped off until skin appeared translucent. E15.5 was the youngest age assessed due to the necessity to capture sufficient cells for RNA-sequencing. Preparation of single cell suspensions and Fluorescence-Activated Cell Sorting (FACS): Tissue was minced and incubated for 30 min at 37°C in digestion media containing RPMI 1640 (Gibco Life Technologies) with 200 units/ml Liberase TL (Roche Applied Science). Up to 1g of tissue was digested per 5 ml digestion media. Tissue was processed using a Medimachine (BD Biosciences) and sterile medicon units (BD Biosciences). Cells were extracted using 1.5-2 ml of RFD solution (24 ml RPMI media, 6 ml FBS, 300 µl 5% DNase I) through a 20 ml syringe with 18-gauge needle. Collected cells were filtered through a 50-micron filter (BD Biosciences). This process was repeated until all the tissue was processed. Cells were spun at 1200 rpm, 4°C for 5 min and resuspended twice in a solution of 1% BSA in PBS and filtered through a 30-micron filter for sorting. Embryos of the same developmental age that were heterozygous for the TRE-H2B-GFP gene but lacked the Dct-rtTA gene were used as negative controls. Cell doublets were excluded from the analysis. Cells were sorted based on GFP expression and SSC-A. Based on these reference sorts, gates were set so that background cells represented less than 10% of sorted cells. RNA Isolation: Cells were lysed in 10-fold TRIzol reagent (w/v), phases were separated by addition of 0.2X volume of chloroform, the aqueous phase was combined with an equal volume of 70% ethanol and applied to a RNeasy Micro column (Qiagen) and processed as per the manufacturer's instructions. Paired-end sequencing libraries were prepared using 1 μg of purified RNA following the mRNA-Seq Sample Prep Kit according to the manufacturer's instructions (Illumina). RNA libraries were prepared for sequencing using standard Illumina protocols