Lambda exonuclease reaction buffer

Lambda exonuclease (λ exo), Exonuclease I (Exo I), Vent DNA polymerase (Vent), Lambda Exonuclease Reaction Buffer (67 mM Glycine-KOH (pH 9.4 @ 25°C), 2.5 mM MgCl₂, 50 μg/ml BSA) and ThermoPol Reaction Buffer (20 mM Tris–HCl, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄ and 0.1% Triton X-100, pH 8.8 @ 25°C) were purchased from New England Biolabs (MA, USA). Taq DNA polymerase (Taq), Pfu DNA polymerase (Pfu) and dNTPs were purchased from Tiangen Biotech Co. (Beijing, China). DNA strands were synthesized and purified by HPLC (Sangon Biotech Co., China). The sequences of all the probes and targets that have been studied in this work are summarized in Supplementary Table S1. DNase/RNase free deionized water purchased from Tiangen Biotech Co. (Beijing, China) was used with all the experiment.

Following template DNA barcoding and purification, the non-transcribed DNA strand was selectively degraded using Lambda exonuclease so that sequencing libraries are amplified exclusively from the transcribed DNA strand, which is guaranteed to correspond to the transcribed RNA sequence. This ensures that TECdisplay measurements are not confounded by heteroduplexes, which may be present in the DNA template preparation. Lambda exonuclease reactions were prepared on ice in 200 μl thin-walled PCR tubes by adding 5.56 μl of 10X Lambda exonuclease Reaction Buffer (NEB) to the 50 μl purified DNA samples, pipetting to mix, adding 0.5 μl of Lambda exonuclease (NEB), and pipetting to mix again. Samples were incubated at 37°C on a thermal cycler block for 5 minutes, and then at 75°C for 10 minutes to heat-inactivate Lambda exonuclease.