GSM5241467: Anagen rep1; Mus musculus; RNA-Seq
FACS isolation of HFSCs from the back skin of control and PRC2 iKO mice was performed as following. The back skin from adult mice was collected. The adipose layer from the dorsal side was scraped off and the scraped skin sample was washed with 1x PBS prior to incubation with 0.25% Trypsin/EDTA (Corning Cellgro) at 37C for 1 hour. After incubation, the epidermal cells, including the HFSCs, was scraped off from the trypsinized skin into the plate. 25 mLs of E-media was added to the cell suspension and was strained through 40µm filters and was washed twice with 1x DPBS. The cells were stained with 1:200 PerCP-Cy5.5-Sca1 (Thermo Fisher Scientific), 1:200 FITC-α6-integrin (Thermo Fisher Scientific), 1:100 APC-EpCAM (Biolegend) and 1:20 Alexa700-CD34 (Biolegend) in staining buffer (HBSS + 2% Fetal Bovine Serum) for 30 minutes on ice and then washed twice with 1x DPBS before cell sorting. HFSCs, was sorted as EpCAM(+), Sca1(-), α6-integrin(high) and CD34(+).FACS-purified cells were collected directly into RLT Plus buffer (QIAGEN), and total RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN). 10ng of total RNA were reverse transcribed and amplified using the Ovation RNA-seq System V2 (NuGEN). Libraries were constructed from 100ng of sonicated cDNA (Covaris) using the Ovation Ultralow system V2 (NuGEN).  The concentration and quality of the libraries were determined using Qubit (Invitrogen) and Bioanalyzer (Agilent). Constructed RNA-seq libraries were sequenced at GENEWIZ on the Illumina HiSeq platform, obtaining 150 nucleotide paired-end reads.