GSM4609868: Aff4-cko-3; Mus musculus; RNA-Seq
For each RNA preparation, skin was isolated from the entire back of 1 mouse at age P6.  The skin was washed briefly with cold PBS, the subcutaneous fat was peeled away with forceps, and the skin was cut in half.  Mesenchyme was then largely removed via heat shock so as to enrich for epithelium (epidermis and hair follicles).  In brief, the two skin pieces were plunged into a beaker of PBS at 60°C and incubated for 30 seconds.  The skin pieces were then transferred to a beaker of cold PBS (on ice) and submerged for 1 min with slow, gentle stirring by hand (with forceps).  The skin pieces were next transferred to a dry petri dish, flattened with the dermal sides down, and grasped at the edges of the epithelial and dermal compartments with separate forceps.  The epithelium and mesenchyme were peeled apart, and the epithelial tissue was snap-frozen using liquid nitrogen.  The frozen tissue was ground to a powder using a pre-cooled mortar and pestle.  RNA was then isolated from the powdered tissue using the RNeasy Mini Kit plus QIAshredder (Qiagen).  To eliminate contaminating DNA, the RNA preparations were treated with TURBO DNAse (2 units per 2 µg of RNA prep; Ambion/Invitrogen) at 37°C for 30 min.  The DNAse was then inactivated using EDTA-Na2 (added to a final concentration of 15 mM) and heat (65°C for 10 min).  A 1/10th volume of 0.3 M sodium acetate was added, and the RNA was ethanol precipitated, dried, and resuspended in RNAse-free water. RNA-seq libraries were prepared using DNAse-treated RNA (1 µg per library) and the Illumina TruSeq Stranded Total RNA Ribo-Zero H/M/R Gold Kit.  Each library underwent ribosomal depletion according to the kit's instructions, was constructed using indexed adapters, was quantified using the KAPA Complete Library Quantification Kit, and was confirmed for quality using an Agilent 2200 TapeStation. The 12 indexed libraries were pooled and sequenced using 2 lanes of an Illumina HiSeq 2500, V4 chemistry, and 50-bp paired-end reads.
GEO Accession
GSM4609868