HDFn-KLF4_dNp63a-d3-RNAseq-1
HDFn cells were grown in Medium 106 (Thermo Fisher Scientific, M-106-500) to 33-66% confluency in 6-well plates and spinfected (1 hour at 2000 rpm) with KLF4 + Np63 lentivirus (1X copy number of each). The spinfection procedure was repeated 24 hours later and fresh media was added at 48 hours. Cells were grown for 3 days after the initial infection, collected by trypsinization, and frozen at -20C. Total RNA was isolated for RNA-seq analysis from the cell pellet using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, AM1931). Samples were treated with DNase I for 20 minutes at room temperature. RNA-seq library preparation and sequencing was performed at the Vanderbilt VANTAGE core. Stranded RNA-seq libraries were prepared from total RNA using poly-A enrichment and the TruSeq RNA Library Preparation Kit (Illumina). Libraries were sequenced on an Illumina NovaSeq 6000 or NextSeq 500 using a paired-end 150 bp protocol.