https://doi.org/10.5061/dryad.5hqbzkhct

We used CRISPR to inactivate mutant KRAS and STAT3 in PANC1 (KRASG12D) pancreatic cancer cell line. Gene expression analysis of KRAS intact vs. knockout cells identified sets of genes involved in protein synthesis, cell differentiation, and metabolic processes, while the expression of MAPK/ERK target genes remained unperturbed.

Methods

Cell line construction

For CRISPR/Cas9-mediated knockouts, we used sgRNAs specific for KRAS and STAT3. Knockout efficiency was measured by Western blotting. Multiple technical replicates were obtained for each CRISPR targeted gene. Genomic DNA from clonal cell lines was collected using QiaAmp DNA Mini Kit (Qiagen). PCR products for sequencing were amplified using Taq DNA polymerase (Invitrogen) and cloned into a pBluescript vector. At least 10-15 bacterial colonies were sequenced per cell line. For RNA isolation, cells were harvested with TRIzol reagent (Invitrogen). Cells were maintained in DME medium supplemented with 5% fetal bovine serum (FBS).

 RNA sequencing

RNA sequencing of tumor cells with basic bioinformatics and statistical analyses were performed by Novogene Corp. The FPKM counts represent fragments per kilobase of transcript per million mapped reads.

Funding

National Cancer Institute

Description of the data and file structure

Data are presented in FPKM format. Data include parental intact (Pa1), KRAS knockout (PaKKO), STAT3 knockout (PaSTAT3KO), and KRAS/STAT3 double knockout derivatives (PaDKO).