T4 dna ligase

The bead-nuclei mixture was again pelleted at 2500g for 60 s, and resuspended in 30 μL H₂O, 20 μL biotinylated bridge-adaptor (see Ma et al [23 (link)] for sequences and adaptor preparation), 20 μL blunt bridge-adaptor, 10 μL 10× T4 DNA Ligase Buffer with ATP, 10 μL polyethylene glycol (PEG)-4000 (Thermo), 5 μL 10 % Triton-X100, and 5 μL T4 DNA Ligase (5 U/μL; Thermo). This mixture was incubated at 16 °C overnight to ligate T-tailed biotinylated bridge adapters to the termini of A-tailed, digested chromatin. Following incubation, the reaction was stopped by adding 5 μL 10 % SDS. The bead-nuclei mixture was then pelleted at 2500g for 60 s and resuspended in 300 μL H₂O. To remove excess unligated adapter, 250 μL 20 % PEG in 2.5 M NaCl was added to the mixture, which was incubated at room temperature for 5 min, collected via DynaMag, and washed once with 80 % ethanol. Beads were then resuspended in 200 μL H₂O and purified further using 0.8 volumes of 20 % PEG in 2.5 M NaCl as above, to further remove unligated adaptors.

The integrity of the single-stranded 3′-CCA ends of the in vitro transcribed tRNAs was tested with a fluorescently labeled RNA/DNA stem loop oligonucleotide (Supplementary Table 3) ligated to the tRNA with 2.5 U T4 DNA ligase (Thermo Fisher Scientific) in 5 µL T4 DNA ligase buffer with 15% (v/v) DMSO overnight at 16 °C. tRNAs with ligated hairpin oligonucleotide and intact CCA termini were separated from the bulk tRNAs on denaturing PAGE. RNAs were visualized by fluorescence or SYBR^TM Gold Nucleic Acid Stain. The approach visualizes only tRNAs with intact CCA-3′ ends and isoacceptors with aberrantly in vitro synthesized 3′ ends or truncated CCA ends in vivo remain invisible^{61 (link)}.

cDNA from Jurkat cells was used to amplify the extracellular domain of the human TRAIL (aa 114-281). Total RNA was obtained from 1×10⁶ cells and converted in cDNA using RevertAid H Minus Reverse Transcriptase (Thermo Scientific) and oligo(dT)18 primer. The resulting cDNA was subsequently PCR amplified with the Phusion High-Fidelity DNA Polymerase (Thermo Scientific) using the primers sTRAIL_F and sTRAIL_R (Table 1) and the 550 bp PCR fragment was gel-purified, digested with EcoRI (Thermo Scientific) and ligated with T4 DNA ligase (Thermo Scientific) at the EcoRI restriction site of pCI.Neo plasmid (Promega), generating the pCI.Neo.sTRAIL plasmid.
The coding sequence of the Fibritin foldon domain, of the T4 Bacteriophage, was humanized using Sequence Manipulation Suite [27 (link)]. Both Foldon_F and Foldon_R ssDNA oligos were hybridized and ligated into the NheI site of pCI.Neo.sTRAIL, with T4 DNA ligase (Thermo Scientific). The resulting plasmid, named pCI.Neo.sfTRAIL, was sequenced to confirm the correct fusion gene structure. Escherichia coli DH5α cells (New England Biolabs, USA) were used for cloning experiments and plasmid propagation. Bacterial strains were routinely grown at 37°C in Luria-Broth (Sigma-Aldrich) or LB-agar (Sigma-Aldrich) containing medium and supplemented with 100 μg/mL ampicillin (Sigma).