RNA-Seq Analysis of Kras Mutant Lung Tumors

Distinct Kras^G12D and Kras^G12DSetd2^–/– mouse lung tumors were dissected and minced into pieces in Trizol (Thermo Fisher Scientific). The minced tumor tissues were then put in Lysing Matrix D tubes (MP Biomedicals) in Trizol and homogenized by FastPrep-24 homogenizer (MP Biomedicals). Total RNA was extracted and cleaned up using RNeasy Mini Kit (Qiagen). Library preparation and sequencing were performed by the Integrated Genomics Operation Core Facility at MSKCC. After RiboGreen quantification and quality control of Agilent BioAnalyzer, 6–15 ng of total RNA underwent amplification (12 cycles) using the SMART-Seq V4 (Clontech) ultra low–input RNA kit for sequencing. In total, 10 ng of amplified cDNA was used to prepare Illumina HiSeq libraries with the Kapa DNA library preparation chemistry (Kapa Biosystems) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4,000 in a 50 bp/50 bp paired end run, using the TruSeq SBS Kit v3 (Illumina). On average, 60 million paired reads were generated per sample, and the percentage of mRNA bases was 73% on average. Raw reads were trimmed and filtered for quality using Trimmomatic (53 (link)). Processed reads were then aligned against the mm10 version of the mouse genome using STAR (60 (link)). For each RefSeq annotated gene, reads overlapping with exon regions were counted using HTSeq (61 (link)). Gene-level differential expression analysis was conducted using DESeq2 (62 (link)).
Differentially expressed genes detected by RNA-Seq (FDR < 0.05) were subjected to GSEA using the JAVA GSEA 3.0 program (63 (link)). The gene sets from the Molecular Signature Database (MSigDB) — including c2 (curated gene sets), c5 (GO gene sets), and c6 (oncogenic signatures gene sets) — were used for the analysis. The composite PRC2 signature was generated by merging the published PRC2 modules in liver cancer (20 (link)), MPNST (21 (link)), hESCs (25 (link)), hematopoietic stem cells (26 (link)), and neural progenitor cells (27 (link)). The KRAS signature was generated by merging the gene sets from MSigDB, including KRAS.600_UP.V1_UP, KRAS.600.LUNG.BREAST_UP.V1_UP, KRAS.BREAST_UP.V1_UP, KRAS.LUNG_UP.V1_UP, and KRAS.KIDNEY_UP.V1_UP. The PTEN_DN_UP signature was generated by merging PTEN_DN.V1_UP and PTEN_DN.V2_UP data sets from MSigDB.