Preparation of 13C-labeled Human snRNAs for NMR Analysis

For in vitro transcription of human ¹³C-labeled U1 snRNA, a cDNA fragment encoding U1 snRNA was amplified by PCR from random primed HeLa cDNA library and inserted into the EcoRI/XhoI sites of plasmid pBluescript II KS(+) (Agilent Technologies, Inc.). ¹³C-labeled U1 snRNA was transcribed in vitro from SpeI-linearized plasmid (1 μg) using Megascript T3 kit (Thermo Fisher Scientific). To transcribe ¹³C-labeled U2 snRNA, a cDNA fragment encoding human U2 snRNA was amplified from random primed TK6 cDNA library by PCR, inserted into HindIII/XhoI sites of pcDNA3.1(+) vector using a GeneArt Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific) and used as a template DNA for the transcription after linearized with XhoI (1 μg). For RNase T1 digestion, RNA was synthesized using Guanosine-¹³C₁₀ 5′-triphosphate, whereas for RNase A digestion, cytidine-¹³C₉ 5′-triphosphate and uridine-¹³C₉ 5′-triphosphate were used instead of the respective 5′-triphosphate reagent that contained carbons with natural isotope distribution. The ¹³C-labeled U1 or U2 snRNA was precipitated in ethanol, solubilized in nuclease-free water and then purified further by reversed-phase LC as described above. The ¹³C-labeled 28S, 18S and 5.8S rRNA were synthesized as described (44 (link)). Primer sequences used for PCR are listed in Supplementary Table S2.