NextSeq 500 paired end sequencing; Influence of Slug/SNAI2 on global gene expression in CRC cells and tumors
HT-29 cells were provided by Richard Camalier (National Cancer Institute, Bethesda, MD, USA). For the genetic Slug model, HT-29 CRC cells were transfected using Lipofectamine and Plus reagent, Invitrogen. Y. Tony Ip, University of Massachusetts Medical School, Worcester, MA, USA generously provided the pCDNA3.1-SLUG plasmid encoding hu-man Slug and the control plasmid. PGL3-OPN (-213)-luc and PGL3-OPN (-1206)-luc con-taining the human proximal and distal OPN promoter, respectively, were a kind gift from Gerhart Ryffel and were provided by Addgene, Waterdown, MA, USA (plasmids # 31106 or 31107). For Tumor Xenograft, Two million cells were injected into the right flank of athymic 6 weeks old female NMRI-Fox 1nu mice (Taconic, Rensselaer, NY, USA). A total of 24 animals were used in this work, divided into 4 groups (WT, Control, Slug1, Slug2). The mice were followed for 24 days from the injection of the cells.  Total RNA was extracted using a mirVana miRNA Isolation Kit from Ambion, Ther-moFisher Scientific,Waltham, MA, USA.  Library preparation was performed using KAPA mRNA HyperPrep kit, Roche