Spliceosome RNA-Seq TT dbr1  (round 2)
250 ng total RNA from purified U2 U5 U6 NTC complex was first subjected to random hydrolysis at 94 C for 3 min in 5  first strand buffer (250 mM Tris-HCl, pH8.3, 375 mM KCl, 15 mM MgCl2) to generate a fragment distribution centered around 60 nts.  For MNase and RNase T1 footprinting libraries, this initial hydrolysis step was omitted because the RNA had already been fragmented will still associated with U2 U5 U6 NTC complex. Following ethanol precipitation, debranched (DBE+) samples were generated by incubating 250 ng RNA with 100 ng recombinant debranching enzyme (DBE) in 100  L DBE buffer (50 mM Tris-HCl pH 7.0, 8 mM MnCl2, 2 mM MgCl2, 25 mM NaCl, 0.01% Triton X-100, 2.5 mM DTT, 0.15% glycerol) for 1 hour at 30 C. Reactions were stopped by adding phenol/chloroform, and the debranched RNA was ethanol precipitated.  For library preparation, 250 ng of branched (DBE-) or debranched (DBE+) RNA was loaded onto a 1.5 mm thick 8% denaturing polyacrylamide gel.  After excision of the region corresponding to the desired fragment length (~10-100 nts), RNA was eluted, phenol extracted and ethanol precipitated. Recovered fragments were then treated with T4 polynucleotide kinase in the absence of ATP to remove 3'-phosphates and subjected to 3'-adaptor ligation, reverse transcription (RT) and cDNA circularization as previously described (Heyer et al., 2015).