3′READS+, a sensitive and accurate method for 3′ end sequencing of polyadenylated RNA

Digestion of poly(A) RNA with a LNA oligo. (A, top) Schematic showing digestion of the poly(A) tail annealed to the T₃₅U₁₅ oligo by RNase H. In theory, the A's hybridized to T's are digested by RNase H, whereas those to U's are not. RNase H digestion is indicated by a lightning symbol. The T₃₅U₁₅ oligo contains a 5′ biotin group that can bind to streptavidin-coated beads. (Bottom) Autoradiography showing digestion products of an RNA molecule containing 60 A's (named A60) by different amounts of RNase H (U/reaction is units per reaction) using the T₃₅U₁₅ oligo. MW, molecular weight markers (sizes indicated). Number of remaining A's in digestion products are indicated, which were calculated based on the molecular weight markers. (B, top) Schematic showing digestion of the poly(A) tail annealed to the T₁₅(+TT)₅ oligos. +T, locked deoxythymidine. (Bottom) Autoradiography showing digestion products of A60 by 1/2 U/reaction of RNase H with different oligos. Number of remaining A's in the digestion products is indicated. (C) Autoradiography showing binding of RNAs with different A's to the biotin-T₁₅(+TT)₅ attached to magnetic beads after washing with buffers containing different concentrations of NaCl and formamide. A60, A15, A10, and A5 have different numbers of consecutive A's and are otherwise the same. (D) Quantification of the amount of A15 and A10 bound to biotin-T₁₅(+TT)₅ relative to A60 in each washing condition based on the data in C.