Sureselectxt mouse exon kit

Next-generation sequencing and data processing of MC-38, TRAMP-C1, CT-26, and EMT-6 cancer cell lines were performed as previously described (Yadav et al., 2014 (link)). Briefly, exome capture was performed using the SureSelectXT mouse exon kit (Agilent). Exome capture libraries were then sequenced on a HiSeq 2000 (Illumina) using the HiSeq sequencing Kit (200 cycles). 60 M (2 × 75 bp) exome reads were sequenced from each cell line. Reads were mapped to the mouse genome (NCBI build 37 or mm9) using GSNAP (Wu and Nacu, 2010 (link)). Only uniquely mapped reads were retained for further analysis. Exome sequencing–based variants were called using GATK (DePristo et al., 2011 (link)) and annotated for effects on transcripts using the variant effector predictor tool (McLaren et al., 2010 (link)). RNA-seq–based variants from the reference mouse genome were called using these criteria: variant allele should be supported by at least two reads, variant allele frequency was ≥4%, and variant allele was not strand-biased (based on read alignment to the genome, Fisher’s exact test, P < 0.05). A vast majority of the variants were supported by exactly two reads, and these accounted for a majority of variants <20% variant allele frequency. As an ad hoc filter, RNA-seq variants with exactly two-read support or <20% variant allele frequency were filtered out.