RNAseq of NTCU-induced mouse lung squamous cell carcinoma

Topical treatment with N-nitrosotris-(2-chloroethyl)urea (NTCU) induces lung squamous cell carcinoma in mouse. The aim of this study is to analyze the expression profile of the mouse NTCU-induced SCC compared to normal lung to identify gene expression changes in the SCC cells. NIH Swiss mice presenting lung SCC and control healthy mice were used for laser capture microdissection (LCM). LCM was performed in a LMD7000 microscope (Leica Microsystems GmbH, Wetzlar, Germany). RNA was extracted using the RNeasy FFPE kit (Qiagen, Hilden, Germany) and libraries were prepared from using the SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian (Clontech Laboratories, Inc., Mountain View, CA, USA). Sequencing was performed using an Illumina HiSeq 2500 generating 50 base pair paired end reads. Reads of low quality were filtered, and adapters trimmed using Cutadapt v1.9.1. Alignment to the reference genome (UCSC mm10) was performed using TopHat v2.0.1, and RNA counts were generated for each gene using the Python package HTSeq v0.6.1, employing the “intersection-strict” overlap mode. DESeq2 was used to normalize and transform the data, examine the relationship between samples (by plotting the first two principal components) and to test for differential expression Overall design: RNAseq of mouse normal lung and lung SCC induced by NTCU