Primary Transcript Enrichment for RNA-seq

A 5 μg quantity of total RNA was used for primary transcript enrichment with our previously published method^{3 (link)}. In brief, the 5′-triphosphorylated RNA species was specifically capped with 3′-desthiobiotin-GTP (New England BioLabs, N0761) by the Vaccinia Capping System (New England BioLabs, M2080S). The RNA was subjected to 3′ adaptor ligation using the same procedure as described above and subsequently enriched with Hydrophilic Streptavidin Magnetic Beads (New England BioLabs, S1421). After washing thoroughly, the RNA was eluted and reverse transcribed to cDNA as described above. The remaining steps were the same as those for library preparation for total RNA SEnd-seq, except that the DNA library was amplified for 15 cycles.