T4 rapid ligation kit

The sequences of all miRNAs were obtained from miRBase (http://www.mirbase.org/), and the optimized pre-miR-34a as well as anti-miR-21, NRF2-siRNA, let-7c, and miR-124 sequences were adopted from our recently study (Ho et al. 2018 (link); Li et al. 2018 (link)). CO-BERA sequences (Table 1) were generated by substituting miR-34a duplexes with target miRNA or siRNA sequences, as illustrated in Fig. 1a, and corresponding coding sequences were synthesized in the pUC57 vector by GenScript Corporation (Piscataway, NJ). Target inserts were released from the plasmids after digestion with EcoRI and PstI (New England Biolabs, Ipswich, MA). Following gel purification using USB PrepEase Gel Extraction Kit (Affymetrix, Inc. Cleveland, OH), each insert was ligated to the EcoRI- and PstI-digested vector pBSTNAV (Ho et al. 2018 (link); Ponchon et al. 2009 (link)) with T4 Rapid Ligation Kit (Thermo Fisher Scientific). The plasmids were then transformed into DH5α competent cells and selected with ampicillin. Colonies were expanded, and CO-BERA expression plasmids were isolated with a Miniprep Kit (Qiagen, Hilden, Germany). All target CO-BERA expression plasmids were confirmed by sequencing analysis (GenScript, Piscataway, NJ).

Plasmid DNA of each fragment was digested with PflMI alone (fragments 2 and 4) or also with SpeI (fragment 1), RsrII (fragment 3) or NotI and RsrII (fragment 5). The pACYC184 vector was digensted with XbaI and EagI to generate cohesive ends that were compatible with SpeI and NotI, respectively, and then gel purified. The digested SHFV cDNA fragments and vector DNA were simultaneously ligated using a T4 Rapid Ligation kit (Thermo Scientific) and the resulting DNA was used to transform XL10-Gold Kan^R Ultracompetent cells (Stratagene). The cells were grown overnight at 37°C in LB media containing chloramphenicol. The full-length SHFV cDNA clones generated were sequenced to insure that no mutations had occurred during cloning.