Gene expression changes in dorsal dermal fibroblasts after beta-catenin deletion in vivo

Goal of this study was to examine gene expression changes upon conditional deletion of beta-catenin at E10.5 in dermal fibroblasts. Method: Skin tissues were treated in 0/25% trypsin for 15 min at 37 degrees C to obtain single cells for FACS sorting and collected in RNA later. RNA was extracted with Karcturus pico pure kit. The RT and amplification was done using Nugen's ovation RNA-seq system V2. Libraries for sequencing were prepared with Illumina TruSeq kit. Transcriptome of FACS sorted E13.5 Engrailed1; RRYFP lineage-marked dorsal dermal fibroblasts was generated next-gen sequencing, in triplicate, using Illumina HiSeq machine. The sequence reads that passed quality filters were analyzed by TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: We mapped about 33-49 million sequence reads per sample and obtained 76-79% of uniquely mapped percentage. We assembled the reads to the mouse ref genome (build mm10). Overall design: E13.5 dermal fibroblast mRNA profiles from En1;RRYFP (n=2) and En1Cre;RRYFP;beta-catenin flox/del (n=3) embryos (from three different litters) were generated by deep sequencing using Illumina Hi Seq