title: Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5
design: The initial template-primer substrate consists of a 41-nt RNA oligonucleotide (5'-AGA UCG GAA GAG  CAC ACG UCU AGU UCU ACA GUC CGA CGA UC/3SpC3/-3’) with Illumina Read1 and  Read2 primer binding sites and a 3' blocking group (three carbon spacer; IDT) annealed to a  complementary 32P-labeled DNA primer to leave an equimolar mixture of A, C, G, or T singlenucleotide  3’ overhangs (Mohr S, et al. (2013) Thermostable group II intron reverse transcriptase fusion proteins and  their use in cDNA synthesis and next-generation RNA sequencing. RNA 19(7):958-970.). Reactions contained ~35 ng IFIT5-bound RNA or 25 ng synthetic miRNA control (miRNAx), 100 nM of the initial template-primer substrate and 2 μM (10 units/μl) TGIRT (TeI4c-MRF RT with a C-terminal truncation of the DNA endonuclease  domain) in 20 μl with final concentrations of 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl at  pH 7.5, 1 mM DTT and 1 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP and  dTTP). Components other than dNTPs were incubated at room temperature for 30 min.  Reactions were initiated by adding dNTPs, incubated at 60°C for 10 min, terminated by adding 5  M NaOH to a final concentration of 0.25 M and incubating at 95°C for 3 min, and then  neutralized with 5 M HCl. The labeled cDNAs were analyzed by electrophoresis in a denaturing  6% polyacrylamide gel, which was scanned with a Typhoon FLA9500 phosphorImager. cDNAs  were electroeluted from gel slices using a D-tube Dialyzer Maxi with MWCO of 6-8 kDa (EMD  Millipore) and ethanol precipitated in the presence of 0.3 M sodium acetate and glycogen carrier  (50 μg; Thermo Scientific). The purified cDNAs were then circularized with CircLigase II  (Epicentre) using the manufacturer’s protocol with an extended incubation time of 5 h at 60°C,  extracted with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated and amplified  by PCR with Phusion-HF (New England Biolabs) using Illumina multiplex (5'- AAT GAT ACG  GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3') and  barcode (5'- CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT  CAG ACG TGT GCT CTT CCG ATC T -3') primers. PCR conditions were initial denaturation  at 98oC for 5 sec followed by 15 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec.  For comparison of wild-type protein purifications, amplification for sequencing was done  using cDNAs pooled from two gel slices of ~55-82 and 84-200 nt, excluding only the 83-nt  cDNA products from template-switches to Read1,2 RNA. For wild-type versus  mutant IFIT5, amplification was performed separately for cDNA size pools a = ~55-82, b = ~84-  150, and c = ~150-230 nt.