CACYBP is required for epidermal homeostasis

RNA-seq was used to define the role of CACYBP in epidermal differentiation by first employing CRISPR/Cas9 to disrupt CACYBP with two independent single guide RNAs (sgRNAs) to create pools of CACYBP-ablated primary human keratinocytes that were immortalized with HPV E6/E7 and then placed onto human dermis and grown for three days at the air-liquid interface to generate organotypic skin tissue. Overall design: Total RNA was then isolated from human skin organoids using QIAshredder (Qiagen) and Trizol (ThermoFisher) followed by DNA removal with the TURBO DNA-free kit (Ambion) according to the manufacturer's instructions. RNA integrity was verified using an Agilent 2100 Bioanalyzer. RNA-Seq libraries were prepared with the mRNA Seq Sample Prep Kit (Illumina, Inc.) as recommended by the manufacturer. 75 bp paired-end sequencing reads were obtained using the Illumina HiSeq platform.