RNA-seq was performed on in vitro and in vivo cell and tumor samples. Specifically, we performed RNA-seq on the following in vitro samples from parental and derivative cell lines: (1) PANC1 and PATU8902 cells with SMAD4 gene-deletion via CRISPR-Cas9 using 2 SMAD4-targeting sgRNA or a control sgRNA, and (2) HPAC and PANC0327 cells with restored SMAD4 expression or expression of Luciferase as negative control. Each parental or derivative cell line was treated either with vehicle (1mg/mL BSA, 4mM HCl) or 10ng/mL TGFß for 24 hours. For in vivo conditions, we performed RNA-seq on the following tumors: (1) matched subcutaneous xenograft tumors from isogenic Lac-Z or SMAD4-expressing HPAC cells harvested at ~300mm3, (2) matched xenograft tumors formed from PATU8902 cells expressing a doxycycline-inducible shSMAD4 shRNA, or its corresponding seed control shRNA (shSMAD4 c7-9), and (3) micro-dissected metastatic tumor from isogenic PATU8902 cells with SMAD4-WT or SMAD4-null genetic status. To obtain matched xenograft tumors formed from PATU8902 cells with dox-inducible shSMAD4, we generated 4 PATU8902 derivative lines, expressing dox-inducible shSMAD4_1, shSMAD4_2, or their corresponding seed control hairpins. Xenograft tumors were first formed in the absence of shRNA knockdown, and when tumors reached ~100mm3, shRNA was induced for 6 days with doxycycline diet (625ppm for 0.6-2 mg/kg in diet) prior to tumor harvest. Snap frozen xenograft tumors were transitioned in RNAlater-Ice (Life Technologies) at -20°C overnight prior to RNA extraction using RNeasy Plus Mini kit (Qiagen). Metastatic tumors from isogenic PATU8902 cell pairs with SMAD4-WT or SMAD4-null genetic status were micro-dissected from mouse lungs embedded in OCT compound (Tissue Tek), and total RNA was extracted using RNeasy Micro kit (Qiagen). Libraries were prepared by tagmentation (Nextera XT DNA Sample Preparation Kit, Illumina) using index primers (Nextera XT Index kit, Illumina) to facilitate multiplexing
GEO Accession
GSM4458573