Human colon total rna

Oligonucleotides (templates and synthetic microRNAs) were purchased from Biomers (Germany). The sequences were purified by high-performance liquid chromatography (HPLC) and checked by matrix-assisted laser desorption/ionization mass spectrometry. Templates were designed according to the rules previously described (23 , 25 (link)). All template sequences (aT, pT, and rT) were protected from the degradation by the exonuclease by 5′ phosphorothioate backbone modifications. A 3′ blocking moiety (phosphate group for aT, pT, and cT and quencher for rT) was used to avoid nonspecific polymerization. Table S1 recapitulates all the sequences used throughout the study. Nb.BsmI and Nt.BstNBI nicking enzymes, Vent(exo-) DNA polymerase, and bovine serum albumin (BSA) were purchased from New England Biolabs (NEB). A 10-fold dilution of Nt.BstNBI was prepared by dissolving the stock enzyme in diluent A (NEB) supplemented with 0.1% Triton X-100. The exonuclease ttRecJ was expressed and purified by chromatography according to a previously published protocol (47 (link)). The enzyme was stored at 1.53 μM in diluent A + 0.1% Triton X-100. All the proteins were stored at −20°C. Human colon total RNA (Thermo Fisher Scientific) was aliquoted at 13 μg/ml and stored at −20°C.