Primary Transcript Enrichment Protocol

Primary transcripts were enriched following a protocol adapted from a previously published method^{8 (link)}. 5 μg of total RNA was mixed with 5 μl of 10× VCE Buffer (New England BioLabs, M2080) in a total volume of 50 μl, incubated for 2 min at 70 °C, and then placed on ice. 5 μl of 3’-Desthiobiotin-GTP (New England BioLabs, N0761) and 5 μl of Vaccinia virus Capping Enzyme (New England BioLabs, M2080) were added to the reaction and incubated at 37 °C for 30 min. After purification with 1.5× RNAClean beads, the capped RNA was eluted and subjected to 3’ adaptor ligation as described above. The RNA was cleaned twice with 1.5× RNAClean beads and then enriched with Hydrophilic Streptavidin Magnetic Beads (New England BioLabs, S1421). After washing thoroughly four times with Binding Buffer (10 mM Tris‐HCl pH 7.5, 2 M NaCl, 1 mM EDTA) and three times with Washing Buffer (10 mM Tris‐HCl pH 7.5, 0.25 M NaCl, 1 mM EDTA), the RNA was eluted with 26 μl of Biotin Buffer (10 mM Tris‐HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA, 1 M biotin) and incubated at 37 °C for 25 min on a rotator. Then 14 μl of Binding Buffer was added and incubated for another 4 min. The RNA was cleaned with 1.5× RNAClean beads and eluted in 12 μl of H₂O. The 5’ capped and 3’ ligated RNA was reverse transcribed by the maturase as described above.