GSM7147049: HF-1 [RNA-seq]; Mus musculus; RNA-Seq
P60 TNC-CREER; tdTomato back skins were collected and subjected to 0.25% trypsin digestion for 60 minutes at 37ºC with 50 rpm shaking. 1:100 SCA1-Cy7 (Biolegend), 1:100 α6-integrin -FITC (Biolegend) and 1:500 EPCAM-APC (Biolegend) for 30 minutes on ice and washed twice with 1x DPBS before cell sorting. Touch dome cells (TD) was sorted as EPCAM (+), SCA1 (+), integrin α6 (+) and TOMATO (+). IFE were sorted as EPCAM (+), integrin α6 (+), SCA1 ( +) and TOMATO (-). Hair follicle cells were sorted as EPCAM (+), integrin α6 (+), SCA1(-) and TOMATO (-). FACS-purified cells were collected directly into RLT Plus buffer (QIAGEN), and total RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN). To generate RNA-seq libraries, 50ng of total RNA was subjected to Poly(A) selection using Universal Plus mRNA-Seq with NuQuant (TECAN). Following elution, mRNA was subjected to fragmentation at 94ºC for 8min. First strand, second strand cDNA synthesis, end repair, adapter ligation and amplification were carried out using Universal Plus mRNA-Seq with NuQuant (TECAN) and by following manufacturer's instructions.  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.