Sephadex g50 columns

Unmodified and fluorescein amidite (FAM) labelled RNA and DNA oligonucleotides were obtained PAGE-purified from IBA (Göttingen) or MWG Eurofins (Munich). The sequences of labelled and unlabelled oligonucleotides used in this study are listed in Table 1. Guide strands were either ordered in a 5’-phosphorylated form or phosphorylated using unlabelled ATP (Fermentas). For cleavage assays, target strands were 5’-phosphorylated using [γ-³²P] ATP (Perkin and Elmer). Phosphorylated oligonucleotides were purified using Sephadex G50 columns (GE Healthcare). Ds DNAs were generated by incubating equimolar concentrations of guide and target strands in hybridization buffer (15 mM Hepes, pH 7.4, 50 mM KCH₃COOH and 1 mM MgCH₃COOH) for 5 min at 95°C, and subsequently slowly cooled down to 37°C. Success of hybridization was tested using 20% native PAGE followed by visualization via the FAM label.

Sequencing reactions were performed in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA), and the same set of PCR primers. Each sequencing reaction was carried in a total volume of 10 μL containing 1 μL of purified PCR product, 0.5 μL of Big Dye^® Terminator v3.1 Ready Reaction Mix (PE Applied Biosystems), 1× sequencing buffer (PE Applied Biosystems), and 3.6 pmol of F or R primer, and the remaining volume consisted of ultrapure water. The sequencing products were purified with Sephadex G50 columns (GE Healthcare) and analyzed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems, Warrington, UK). Sequences were edited using the Sequencher software v. 5.2.4 (Genes Codes Corporation, Ann Arbor, MI, USA) and the primer regions were removed.

Sequencing was performed in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and the same set of PCR primers. The sequencing products were purified using Sephadex G50 columns (GE Healthcare, Chicago, IL, USA) and analyzed in an Applied Biosystems 3130 DNA Analyzer (PE Applied Biosystems). Sequences were edited in Sequencher v.4.9 software (Genes Codes Corporation, Ann Arbor, MI, USA) and the primer regions removed. Sequences from GenBank or those previously processed in the Systematics Molecular Laboratory (FSP) were also included to serve as references in the analyses. The COI gene sequences were aligned by nucleotide using the muscle algorithm [51 (link)], implemented in SeaView [52 (link)], and then by amino acid using TranslatorX [53 (link)].

For some specimens, it was problematic to obtain the entire mitochondrial DNA using Illumina sequencing technology only. Consequently, we obtained small fragments of certain portions of the mitochondrial genome to complete the circular DNA molecule. In these situations, we amplified the target DNA using primers that were developed for specific regions (electronic supplementary material, table S5). PCR products were electrophoresed in 1.0% TBE agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium Inc., Hayward, USA). Sanger sequencing reactions [38 (link)] were carried out in one direction using ABI Big Dye Terminator Kit v.3.1 (PE Applied Biosystems, Warrington, England). Sequencing reactions were purified in Sephadex G50^® columns (GE Healthcare), analysed on an ABI Prism 3130—Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA), and edited using Sequencher^® for Windows v. 5.1. Sanger DNA fragments were assembled to the mitochondrial genome obtained using Illumina sequencing technology to complete the circular molecule.

The short RNAs as2b, s2b, aslam and slam were ordered from IBA or Biomers. ICAM-1 in vitro transcript (ICAM-1-IVT) was transcribed from plasmid pG-si2b (+) using T7 RiboMAX™ Express Large Scale RNA Production System (Promega) and afterwards purified using phenol-chloroform extraction and ethanol precipitation. Aslam-FAM and slam, as well as as2b and s2b or as2b-FAM and s2b were annealed in order to obtain siRNA-like duplexes. All nucleic acid sequences used in the present study are listed in Table 1.
As2b as the guide strand was phosphorylated at the 5’-end with unlabeled ATP (Fermentas), while ICAM-1-IVT and s2b were 5’-phosphorylated with [γ-³²P] ATP (Hartmann, PerkinElmer) where appropriate. The modified RNAs were purified by Sephadex-G50 columns (GE Healthcare), phenol-chloroform extraction and ethanol precipitation. Complementary RNAs were annealed using equimolar amounts of both strands in siRNA annealing buffer (15 mM HEPES pH 7.4, 50 mM KCH₃COOH, 1 mM MgCH₃COOH). The two strands were incubated for 3 min at 95°C and slowly cooled down to room temperature. The integrity of the hybridized siRNA was tested using native PAGE-analysis visualized by using radioactive- or FAM-labelled RNAs or by staining with Stains-All (Sigma-Aldrich).

CCL25 and CCR9 DNA fragments were cloned from human thymus cDNA (Biochain, Hayward, CA) using primers 5′-ATGAACCTGTGGCTCCTG-3′ and 5′-TAACAGGCAGGAATGACTC-3′ (CCL25) and 5′-ATGACACCCACAGACTTC-3′ and 5′-GAGGGAGAGTGCTCCTG-3′ (CCR9). The fragments were isolated by agarose gel electrophoresis, labeled with ³²P-cytidine 5′-triphosphate (GE Healthcare, Piscataway, NJ) using the NEBlot kit (New England Biolabs, Ipswich, MA) and purified on Sephadex G-50 columns (GE Healthcare, Piscataway, NJ). Normal tissue mRNAs were purchased from Ambion (Austin, TX), Biochain (Hayward, CA), Origene (Rockville, MD), and Cybrdi (Frederick, MD). RNAs were subjected to agarose gel electrophoresis (2 µg/lane) and blotted to nylon membranes using the Northernmax kit (Ambion, Austin, TX). The membranes were prehybridized with Ultrahyb (Ambion, Austin, TX) at 42°C for 1 hr, and the radiolabeled probes were boiled and added (~5 × 10⁶ cpm/blot). After overnight hybridization at 42°C, the probes were removed and the blots were rinsed successively with 2x SSC 0.1% SDS and 0.1x SSC 0.1% SDS at 42°C. Blots were exposed to Hyperfilm MP (GE Healthcare, Piscataway, NJ) which were developed with a Mini-Med 90 (AFP, Elmsford, NY).

Sequencing reactions proceeded in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and Applied Biosystems 3130 DNA Analyzer (Applied Biosystems). Sequencing reactions were carried out with the same set of PCR primers. The sequencing products were purified using Sephadex G50 columns (GE Healthcare) and analysed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems). Sequences were edited in Sequencher v.4.9 software (Genes Codes Corporation, Ann Arbor, MI, USA), and the primer regions removed. In addition to these novel sequences, 162 sequences from GenBank were also included, to serve as references in the phylogenetic and species delimitation analysis. The COI gene sequences were aligned first by nucleotides using the Muscle algorithm [48 (link)] implemented in SeaView [49 (link)], and then by amino acid using TranslatorX [50 (link)].