TP53 and TP63 modulation of Squamous PDAC

Based on the association of TP53 mutation and upregulated TP63 expression in the squamous subtype, we used cell lines derived from genetically engineered mouse models of PDAC (KRAS Trp53fl/+ and KRAS Trp53fl/+ Trp63fl/fl) to begin to unravel the functional consequences of these events in defining squamous tumours. RNA-Seq libraries were generated using TruSeq Stranded Total RNA (Part no. 15031048 Rev. D April 2013) kits, using on a Perkin ElmeraTMs Sciclone G3 NGS Workstation (Product no. SG3-31020-0300). Ribosomal depletion step was performed on 1 ug of total RNA using Ribo-Zero Gold prior to a heat fragmentation step aimed at producing libraries with an insert size between 120-200 bp. cDNA was then synthesized from the enriched and fragmented RNA using InvitrogenaTMs SuperScript II Reverse Transcriptase (Catalog no. 18064) and random primers. The resulting cDNA was further converted into double stranded DNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintain the strandedness of the library. Following 3aTM adenylation and adaptor ligation libraries were subjected to 15 cycles of PCR to produce RNA-Seq libraries ready for sequencing. Prior to sequencing, exome and RNA-Seq libraries were qualified and quantified via CaliperaTMs LabChip GX (Part no. 122000) instrument using the DNA High Sensitivity Reagent kit (Product no. CLS760672). Quantification of libraries for clustering was performed using the KAPA Library Quantification Kits For Illumina sequencing platforms (Kit code KK4824) in combination with Life Technologies Viia 7 real time PCR instrument. All libraries were sequenced using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3 - HS.