Sephadex g 25 spin column

The coding sequences of human NSUN3, ABH1 or FTO were cloned into a pQE80 derivative encoding an N‐terminal His₁₄‐MBP‐tag (Weis et al, 2014) and the CDS of MTIF2 or TUFM into a pQE80 derivative encoding a C‐terminal His₁₀‐tag (Mingot et al, 2004). The ABH1 D233A and R233A, and NSUN3 C265A mutants were generated by site‐directed mutagenesis (Haag et al, 2015b). Recombinant proteins were expressed in Escherichia coli (DE3) Rosetta pLysS (NSUN3, ABH1) or (BL21) Codon Plus (MTIF2, TUFM) cells and details of protein purification are given in the Appendix Supplementary Methods. Mt‐tRNA^Met, mt‐tRNA^Glu, mt‐tRNA^Pro, tRNA_i^Met and tRNA_e^Met sequences were generated by recursive PCR as described (Müller et al, 2013) using four overlapping oligonucleotides each and cloned into a pQE vector derivative lacking an internal T7 promoter. The CCA tail and a BsaI restriction site were added at the 3′ end of the tRNA gene and the forward primer contained the sequence of the T7 promoter. Point mutations were introduced by site‐directed mutagenesis. For in vitro transcription, 500 ng of BsaI‐linearised plasmid were incubated with 1 mM NTPs, T7‐RNA polymerase, 1× transcription buffer (Thermo) and RiboLock (Thermo) for 1 h at 37°C. After transcription, samples were treated with DNase I for 15 min and purified over a Sephadex G‐25 spin column (Roche).

Oligonucleotide probes complementary to mature miRNAs were end-labeled with γ-³²P-ATP using T4 Polynucleotide Kinase (Takara, Dalian, China), and labeled probes were purified using a Sephadex G25 spin column (Roche, Indianapolis, IN, USA). Small RNA was extracted from 10 g of HS or 10 ml of HS decoction. RNA samples were fractionated by PAGE using a 15% denaturing polyacrylamide gel. The RNA was then transferred onto a nylon membrane (Hybond N⁺, Amersham Biosciences, Piscataway, NJ, USA) by electroblotting at 400 mA in 0.5× TBE buffer for 1.5 h. The membrane was cross-linked and dried. A prehybridization step was performed by incubating the membrane with 10 ml of ULTRAhyb-Oligo solution (Ambion, Austin, TX, USA) at 37 °C for 1 h. The radiolabeled probe was added directly to the ULTRAhyb-Oligo solution, and the membrane was incubated overnight at 37 °C with rotation in a hybridization oven. After hybridization, the membrane was washed twice at low stringency in 2× SSC, 0.1% SDS at 42 °C for 10 min. The membrane was wrapped in a plastic wrap and exposed to an X-ray film at −80 °C.