Amicon ultra 0.5 ml 3k filter tubes

Synthesis columns containing oligonucleotides were treated previously with 10% diethylamine (Fisher) in ACN on the synthesizer. Oligonucleotides were cleaved and base-protecting groups were removed with a solution of 1:1 40% methylamine in water/30% ammonium hydroxide for 2 hours at room temperature. Cleaved and deprotected oligos were fully dried under a vacuum. Oligonucleotides containing 2’TBDMS protecting groups were dissolved in 115 μL DMSO (Sigma-Aldrich) at 65 °C. Triethylamine 60 μL (Sigma-Aldrich) followed by Triethylamine-trihydrofluoride 75 μL (Sigma-Aldrich) was added, and the whole solution was incubated for 2.5 hours at 65°C. Deprotected oligonucleotides were cooled and precipitated in a solution of 0.1 M sodium acetate in isopropanol (Sigma Aldrich). After centrifugation, the supernatants were discarded, and the pellets were dried under a vacuum. Dried oligonucleotides were dissolved in 400 μL RNase-free water and desalted using Amicon Ultra 0.5 mL 3K filter tubes (Millipore, Billerica, MA USA), followed by three RNase-free water wash rounds for 15 min at 14000 × g. Finally, desalted oligonucleotides were extracted and diluted in RNase-free water.

Synthesis columns containing oligonucleotides were treated with 10% diethylamine (Fisher) in ACN on the synthesizer. Oligonucleotides were cleaved and base-protecting groups were removed with a solution of 1:1 40% methylamine in water/30% ammonium hydroxide for 2 h at room temperature. Cleaved and deprotected oligos were fully dried under a vacuum. Oligonucleotides containing 2′TBDMS protecting groups were dissolved in 115 μl DMSO (Sigma-Aldrich) at 65°C. Triethylamine 60 μl (Sigma-Aldrich) followed by Triethylamine-trihydrofluoride 75 μl (Sigma-Aldrich) was added, and the whole solution was incubated for 2.5 hours at 65°C. Deprotected oligonucleotides were cooled and precipitated in a solution of 0.1 M sodium acetate in isopropanol (Sigma Aldrich). After centrifugation, the supernatants were discarded, and the pellets were dried under a vacuum. Dried oligonucleotides were dissolved in 400 μl RNase-free water and desalted using Amicon Ultra 0.5 ml 3K filter tubes (Millipore, Billerica, MA USA), followed by three RNase-free water washes, spinning in each case for 15 min at 14 000 × g. Finally, desalted oligonucleotides were dissolved in RNase-free water.