Typhoon fla 7000 fluorescent image analyzer

100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m⁷Gppp-RNA (45 nt) were incubated with 100 mM HEEB (1 M stock in DMSO) with ADPRC (25 μg/ml) in 100 μl of ADPRC reaction buffer (50 mM Na-HEPES pH 7.0, 5 mM MgCl₂) at 37°C for 1 h. 100 μl of DEPC-treated H₂O was then added and acid phenol/ether extraction was performed to stop the reaction (17 (link)). RNAs were precipitated by ethanol, re-dissolved in 20 μl of DEPC-treated H₂O. 5 μl of biotinylated RNAs were kept as input. After HEEB reaction, biotinylated RNAs were incubated with streptavidin bead particles (6 μl, MedChemExpress, catalog: HY-K0208) and 0.4 U/μl of RNase Inhibitor (Takara Bio, catalog: 2313B) at 25°C for 30 min. Beads were washed four times with streptavidin wash buffer (50 mM Tris–HCl (pH 7.4) and 8 M urea), and three times with DEPC-treated H₂O. The biotinylated RNA on the beads was extracted with Trizol LS (Ambion, catalog: 10296010) and chloroform. Input (see above) RNAs and the biotinylated RNA on the beads were analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).

100 ng of spike-in NAD-RNA (38 nt) and 200 ng of m⁷Gppp-RNA (45 nt) were mixed together and incubated with 500 nM NudC (New England Biolabs, catalog: M0607S) in 25 μl of NudC reaction buffer (100 mM NaCl, 50 mM Tris–HCl pH 7.9, 10 mM MgCl₂, 100 μg/ml Recombinant Albumin) at 37°C for 30 min. Product was purified with Trizol LS (Ambion, catalog: 10296010) and analyzed by an 8% polyacrylamide TBE urea gel. Gel was stained by SYBR Gold (Invitrogen, catalog: S11494) and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).

Boronic acid beads (30 μl, Smart-Lifesciences, catalog: SA057005) were washed 3 times with binding buffer (50 mM Na-HEPES, 1 M NaCl, PH 7.8), and then incubated with RNA oligonucleotide in binding buffer in a thermomixer at 37°C for 2 h. Beads were then washed 3 times with wash buffer (50 mM PBS, pH 7.4, 6 M urea); RNA oligonucleotide bound by the beads was extracted with Trizol LS (Ambion, catalog: 10296010)). Purified RNA product was analyzed by an 8% polyacrylamide TBE–urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).

Magnetic streptavidin beads (6 μl, MedChemExpress, catalog: HY-K0208) were washed 3 times with immobilization buffer (10 mM Na-HEPES, 1 M NaCl, 5 mM EDTA) (17 (link)), and then incubated with the purified RNA product from HEEB reaction in immobilization buffer, together with 0.4 U/μl of RNase Inhibitor, in a thermomixer at room temperature for 30 min. After the beads were washed 5 times with streptavidin wash buffer (50 mM Tris–HCl, pH 7.4, 8 M urea) (17 (link)), the biotinylated RNA on the beads was extracted with Trizol LS (Ambion, catalog: 10296010) and chloroform. The upper aqueous layer was further purified with Zymo column (Zymo Research, catalog: R1016). The purified RNA product was analyzed by an 8% polyacrylamide TBE urea PAGE gel. Gel was stained by SYBR Gold and fluorescence was detected by Typhoon FLA 7000 fluorescent image analyzer (GE Life Science).