title: GSM3045724: XPO5_het_rep2; Mus musculus; miRNA-Seq
library_construction_protocol: For the miRNA-seq, total RNA was extracted by Trizol reagent (Thermo Fisher Scientific) using HEK293Tcells and epidermis cells isolated from XPO5 het and KO animals . For miRNA-seq, Total RNA samples from p2-p4 back skin epidermis and HEK293T cells were subject to high efficiency 3′ ligation. Products were resolved on 15% PAGE-urea gels and stained with SybrGold (Molecular Probes S-11494 Carlsbad, California, USA). The region corresponding to ligated miRNAs (46 nucleotides) was excised, gel slices were thoroughly minced, and eluted in HSCB (400 mM NaCl, 25 mM Tris-HCl pH 7.5, 0.1% SDS) overnight at 4°C. Nucleic acids were precipitated in the presence of glycogen carrier via 0.1 volumes sodium acetate and 2.5 volumes ethanol. Pellets were washed in 70% ethanol, and dissolved in the 5′ ligation mix without enzyme. Samples were briefly heated to 70°C, snap chilled on ice, and then enzyme was added. Following ligation reactions cDNA was prepared using Superscript III RT (Invitrogen) according to the manufacturer′s recommendation using 3′ linker-specific RT primer. cDNA libraries served as templates for PCR amplification; amplicons were resolved on 8% native acrylamide gels. Bands of the correct molecular weight were isolated and used for high-throughput sequencing on the Illumina HiSeq2000.