NextSeq 500 paired end sequencing; Dissecting the peculiar RIZ2 oncogenic properties in colorectal cancer: a link with EGF/EGFR axis
Dissecting the peculiar RIZ2 oncogenic properties in colorectal cancer: a link with EGF/EGFR axis
Human colorectal adenocarcinoma cell lines DLD-1,  were provided and grown as previously described [Proto et al., 2017; Notarnicola et al., 2008]. When indicated 48 hours before stimulation, growing cells at 70% confluence were made quiescent using phenol red-free RPMI medium containing 0.1% charcoal-stripped serum (CSS), penicillin (100 U/ml) and streptomycin (100 U/ml). The hEGF (Sigma Aldrich, St. Louis, Missouri, USA) was used at 100 ng/ml. The EGFR tyrosine kinase inhibitor, ZD1839, (Selleckem, Planegg, Germany) was used at 2 �M. The EGF neutralizing antibody (10605-R001; Sino Biological, Beijing, China) was used at 1,5 �g/ml. DLD-1-pEGFP and DLD-1-pEGFP_hRIZ2 cells were counted by the method of optical microscopy in the B�rker chamber. DLD-1-pEGFP_hRIZ2 cells were compared with DLD-1-pEGFP cells. total RNAs concentration was measured using RNA HS kit on a Qubit fuorimeter (Life Technologies, Monza, Italy) while its quality and integrity assessed with the Agilent 4200 Tapestation System (Agilent Technologies, Milan, Italy). For RNA sequencing experiments, indexed libraries were prepared using 500 ng of total RNA as starting material using the Illumina Stranded Total RNA prep Ligation with Ribo-Zero Plus kit (Illumina Inc., San Diego, CA, USA).