Primary mouse keratinocytes - 72 h, low calcium
Primary keratinocytes were isolated from newborn mice and plated under proliferating or differentiating (2.0 mM Ca2 ) conditions in custom keratinocyte media.  Cells were harvested at 72 hours post-calcium treatment and total RNA was isolated by TriZol extraction (Life Technologies).  Poly-A mRNA was enriched using the Dynabeads mRNA purification kit (Life Technologies) and fragmented prior to SuperScript III 1st-strand synthesis system (Life Technologies) and subsequently the NEBNext mRNA Second Strand Synthesis Module (NEB).  RNA-seq libraries were generated according to the manufacturer’s instructions (Illumina) and sequenced using the Illumina HiSeq 2000.