Journal: bioRxiv

Article Title: A robust method for measuring aminoacylation through tRNA-Seq

doi: 10.1101/2023.07.31.551363

Figure Lengend Snippet: Despite optimization attempts, high ligation efficiency could not be achieved for blunt-end ligation. (A) Effect of incubation temperature, time and addition of a phosphatase (rSAP). Using deacylated and gel purified human tRNA as substrate and pre-adenylated l3N as adapter, otherwise following the method in Behrens et al. ( 2021 ). (B) Effect of additives and higher adapter concentration. Using deacylated and gel purified human tRNA as substrate, pre-adenylated l2N as adapter and 4°C, 24 h incubation. An irrelevant well has been crossed out to avoid image splicing. (C) Effect of ligase type. Using the E.coli tRNA-Lys-CCA oligo as substrate, pre-adenylated l1N as adapter and 4°C, 24 h incubation with 20% DMSO. Wells with “+l1N” were added additional none pre-adenylated adapter. Rnl2tr KQ (T4 RNA Ligase 2, truncated KQ) is the standard ligase used for pre-adenylated adapters whereas Rnl2 (T4 RNA Ligase 2) does not require pre-adenylation of adapters.

Article Snippet: 35 ng of the annealed tRNA was transferred to a PCR tube and to this was added 20 pmol annealed adapter:splint partial duplex, 1 μL 10x NEBuffer 2, 2 μL 10x T4 RNA ligase buffer, 4 μL 50% PEG-8000, 1 μL SuperaseIn and 1 μL T4 RNA ligase 2.

Techniques: Ligation, Incubation, Purification, Concentration Assay

Journal: bioRxiv

Article Title: A robust method for measuring aminoacylation through tRNA-Seq

doi: 10.1101/2023.07.31.551363

Figure Lengend Snippet: (A) Ligation test comparing the effect of RNA processing. Similar to , panel E but with two different adapters. (B) The unligated tRNA that appears after tRNA is oxidized with periodate in panel A is refractory to further ligation. The unligated tRNA was gel purified from enough ligation reactions as shown in panel A to setup two new ligation reactions using either l1N pre-adenylated adapter for blunt end ligation or l6Sp for splint assisted ligation. For l1N, ligation was setup with 35 ng tRNA, 20 pmol adapter, 17.5% PEG-8000, 20% DMSO, 1×T4 RNA ligase buffer, 1 μL T4 RNA ligase 2 (truncated KQ) and 1 μL SuperaseIn. For l6Sp, the ligation was setup as described in the charge tRNA-Seq protocol.

Article Snippet: 35 ng of the annealed tRNA was transferred to a PCR tube and to this was added 20 pmol annealed adapter:splint partial duplex, 1 μL 10x NEBuffer 2, 2 μL 10x T4 RNA ligase buffer, 4 μL 50% PEG-8000, 1 μL SuperaseIn and 1 μL T4 RNA ligase 2.

Techniques: Ligation, Purification