GSM5088069: Basal bulge stem cells (B); Mus musculus; RNA-Seq
After sacrification, backskin was harvested from 4 mice. Epidermis was separated from dermis after 1h incubation with 0.25 mg/ml thermolysin. Epidermis was minced and cells were filtered. Cells were stained with anti-CD34 and Itga6 conjugated antibodies. 3 populations were sorted via FACS:  CD34- (non-stem cells 'N'), CD34+/Itga6High (basal bulge stem cells 'B'), CD34+/Itga6Low (suprabasal bulge stem cells 'S'). Total RNA was extracted from the cells with Trizol reagent. RNA from 4 mice per FACS sorted cell population (N, B and S) was pooled. Due to low amount of input material, pre-amplification using the Ovation RNASeq System V2 was performed. Total RNA was used for first strand cDNA synthesis using both poly(T) and random primers, followed by second strand synthesis and isothermal strand-displacement amplification. For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used with 1 ng cDNA input. After validation (Agilent 2200 TapeStation) and quantification (Invitrogen Qubit System) all transcriptome libraries were pooled. The pool was quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection and sequenced on an Illumina HiSeq4000 sequencing instrument with a 2x75bp paired-end read length.