RNAseq transcriptomic signature associated depletion of Evi/Wls in epidermal sheets of mice, expression analysis of epidermal sheets of Evi/Wls knockout and wildtype mice
RNAseq transcriptomic signature associated depletion of Evi/Wls in epidermal sheets of mice, expression analysis of epidermal sheets of Evi/Wls knockout and wildtype mice
Evi floxed mice were crossed with K14-Cre mice to delete Evi gene in epithelial cells. Skin of 21 day old mice was collected from mutants and wildtype animals. After tissue digestion RNA from epidermal sheets was prepared. Three fully independent biological replicates where sampled. RNA was extracted from epidermal sheets of three wildtype and Evi-LOF mice. The poly(A)+ fraction was isolated from each of the six samples with the Dynabeads mRNA Purification Kit (Invitrogen). For each sample, a library for sequencing with a SOLiD4 sequencer was prepared. random primers with a hexamer adapter sequence were annealed to the 3'  end of the mRNA and the first DNA strand synthesized. Following RNA digestion, the DNA was purified using Agencourt RNAClean XP Beads. Random primers were annealed to the DNA for second strand synthesis. Agencourt Ampure XP Bead selection was used to purify and size select the double stranded DNA. The library was amplified and barcoded.