RNA-seq of human patient-derived xenograft pancreatic cancer Panc185 sphere-derived cells with the ISG15 gene edited using CRISPR-CAS9 against control spheres.

ABSTRACT: The goal of the study was to identify genes and pathways that were altered in the absence of the gene Interferon-Stimulated Gene 16 (ISG15) in human pancreatic ductal adenocarcinoma (PDAC) cancer stem cells. Primary adherent cultures established from patient-derived xenograft passaged in mice were established (Panc185). The ISG15 gene was edited using CRISPR-CAS9 technology and 3 commercially available guide RNAs introduced via lentiviral infection. Control cultures were also established using control guide RNAs. Once a polyclonal population was established and ISG15 loss confirmed, cultures were grown as 3D spheres in ultralow attachment plates to achieve a CSC-enriched culture. Following 5 days in culture as spheres, Total RNA was isolated by the guanidine thiocyanate (GTC) method using standard protocols. PolyA+ RNA fraction was processed as in Illumina’s ‘‘TruSeq RNA Sample Preparation v2 Protocol’’. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer’s protocols. RNA-seq data sets were analyzed using the tool Nextpresso.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Bruno Sainz, Jr. 

PROVIDER: E-MTAB-8984 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress