title: Mutant mouse neonate dermis RNA-Seq
design: We performed a double selection of polyadenylated RNA using oligo-dT magnetic beads. Next, the RNA was fragmented with RNA fragmentation buffer and removed free-ions with a G-50 Sepharose spin column. Fragmented mRNA was used as a template to synthesize single-stranded cDNA with SuperScript II reverse transcriptase and random hexamer primers in the presence of RNaseOUT. To prepare cDNA for sequencing, we performed end repair using T4 DNA polymerase and Klenow DNA polymerase, addition of an A base to the 3-prime ends of the cDNA using Klenow Fragment, followed by ligation of adaptors designed for the Illumina sequencing platform. The ligation product was purified by gel electrophoresis and purification of the 175 to 225 base pair region on a 1.5% NuSieve GTG agarose gel using the QIAquick Gel Extraction kit. Finally, we enriched the library with 15 cycles of PCR amplification using Illumina sequencing primers.