title: NextSeq 500 paired end sequencing; DOCK1 gene derived hsa_circ_0020397 RNA is over-expressed in luminal mammary tumors and it is involved in the epithelial differentiation, growth and motility of the neoplastic cell
library_construction_protocol: Breast cancer cell lines were grown in DMEM-F12 medium in the presence of 5% fetal calf serum. To obtain circDOCK1 over-expressing cellular populations, the DNA coding for circDOCK1 was cloned into the pCDNA3 plasmid, which was transfected in the MDA-MB-157 and MDA-MB-231 breast cancer cell-lines using the FuGENE6 (Promega). Neomycin resistant cell clones were selected in the presence of G418 (MDA-MB-157 cells = 400 µg/ml; MDA-MB-231 cells = 500 µg/ml) for two weeks. Approximately 20 G418 resistant clones were isolated from each cell line and the levels of circDOCK1 expression were examined by RT-PCR. At least 3 clones with highest amounts of circDOCK1 RNA expression were expanded from each cell line and used for further experiments. To obtain negative control clones, the cells were transfected with void vectors and at least 3 of G418 resistant clones were isolated and used for determination of basal levels of circDOCK1. To obtain circDOCK1 silencing, CAMA1 cells were infected with lenti-viral particles containing 2 independent circDOCK1 targeting sh-constructs cloned in the plasmid pINDUCER10 (Addgene, Watertown, MA), and the cellular pools obtained the sh constructs were selected in the presence of puromycin (1 µg/ml) for two weeks. Puromycin resistant cell pools were then divided into two groups and they were treated in the absence (as controls) or in the presence of 0.5 µg/ml of doxycycline for four days to induce the shRNAs. Cells with reduced levels of circDOCK1 expression respect to the basal conditions (controls) were expanded and used for further experiments. For the RNAseq analysis of circDOCK1 overexpressing clones and the respective control clones, three paired biological replicates of each cell-line were grown in the presence of G418. For the silencing constructs, three biological replicates of cells were treated for 4 days in the presence or absence of doxycycline. Collection of 5-10 million cells in PDS, and resuspension of the pellet in a buffer. RNA was extracted with the mRNeasy Mini Kit (QIAGEN).  TruSeq Stranded Total RNA Sample Preparation Guide