a, Preparation of FragSeq libraries for sequencing. RNA 5′ and 3′ end chemistry is specifically shown to highlight PNK and nuclease products; when RNA end chemistry is not shown, it denotes any possible end chemistry. Only clonable RNA fragments are shown at and after the size-selection step. Lightning bolts represent the specific ligation events at each end of the RNA fragment. b, Overview of the FragSeq algorithm.

a-e, Data tracks in the UCSC Genome Browser (mm9 mouse genome assembly) showing spliceosomal snRNA U1a (a); data from mouse undifferentiated embryonic stem cell samples (UNDIFF) (b-d) is processed to get cutting scores, which are compared to cutting scores from D5NP cells (e). Ignored sites (Supplementary Note 1) are denoted in (e) as areas for which no data is shown (e.g. the sequence GUG in the Sm region). f, Sequence of U1a, highlighting regions shown in (g) using the same color code; green and yellow subsequences are expected to be single-stranded. g, Cutting scores (blue arrows) from UNDIFF sample (e) superimposed on the known secondary structure. Non-canonical base pairs in interior loops of stem 2 are shown as unpaired. 2′-O-methylated positions are not depicted. SL, stem-loop; IL, interior loop; MBL, multibranch loop. U1a structure is from several sources (Supplementary Note 3).

a-b, Probing results for human U3 purified from HeLa cells¹⁵ (a) and FragSeq cutting scores for mouse U3b (b). c-d, Probing results for human U5 purified from HeLa cells¹⁶ (c) and FragSeq cutting scores for mouse U5 (d). Black arrow shows priming position for primer extension; only bases downstream of the primer were probed in that study (c). Reactivities in (a) and (c) are taken verbatim from ref. 15 and ref. 16, respectively; structures and other annotations were compiled from multiple sources (Supplementary Note 3). 2′-O-methylated positions are not depicted.

Coverage (mean reads per nucleotide) is shown at right for nuclease and control treatments. a, Cutting scores compared to ssRNA regions greater than three bases long (green boxes) for ncRNAs with published structure models (Supplementary Note 3). Regions exist where the in vitro structure of a single, naked RNA is uncertain (olive boxes). SL, stem-loop; Sm, Sm protein binding site; BP, splicing branch-point binding site; Flank, flanking ssRNA region of a nearby motif; IL, interior loop; Hinge, ssRNA region connecting two RNA domains; Kturn, kink-turn RNA motif containing non-canonical base pairs. b, Cutting scores for all long (> 120nt) C/D box snoRNAs considered for follow-up probing. RNAs with an asterisk (*) were chosen for follow-up probing.

a, FragSeq ssRNA cutting scores (bottom, dark blue) and band quantification readouts (SAFA counts) based on the gel resolving 5′-end-labeled probing products. X-axis shows nucleotide numbering; gridlines appear every five nucleotides. Gray nucleotides in sequence show areas that were outside of the reliably quantifiable area on the gel. Parentheses denote Watson/Crick base pairs and dots denote ssRNA. Triangles denote bases where a nuclease can cut: T1, gray triangles at G; RNase A, black triangles for C and red triangles for U. Outlier values were truncated and marked with red zigzag lines. b, Follow-up probing data superimposed on our structure model, with probing enzymes color-coded as in (a). Marginal, weak, moderate, or strong enzyme activity was inferred from manual inspection of the gel and Safa quantification from (a) (Supplementary Note 3). c, FragSeq cutting scores superimposed on the same structure model as (a) and (b). Boxes (green) and the putative region that base-pairs with target rRNA (orange) are highlighted, with the base opposite of the methylated position²⁶ in red. Highlighting is as in (b).