Extended Data Fig. 6: Additional comparisons of libraries made from mirRICH, total RNA, or gel-base size-selected RPFs.

a, A SYBR Gold stained 0.6X TBE 12% denaturing urea–PAGE gel of resolved P1 nuclease digested yeast RNA purified from either Direct-Zol Total RNA extraction or mirRICH small RNA enrichment following purification by sucrose cushion. Roughly 5% of the Direct-Zol extract and 25% of the mirRICH extract was analyzed in this gel. The migration of the 30 nt (light blue) and 40 nt (dark blue) size selection RNA oligos are demarcated (left). Both lanes are from the same gel, and both purification methods were replicated twice. b, As in Extended Data Fig. 3a, cDNA size selection from a OTTR library generated from 40 ng of mirRICH small RNA from (a). Parallel positive control OTTR libraries were synthesized from either 30 or 40 nt size selection RNA oligos to aid in cDNA size selection. The single light blue and dark-blue dots (left) demarcate the cDNA with inserts derived from the 30 nt and 40 nt RNA oligos, respectively. cDNA concatemers from deliberately using more templates in OTTR, for example ~80 nt insert, are demarcated by two dark blue dots. monosome cDNA size selection area was demarcated by the bottom two black lines (right), and the disome cDNA size selection by the top two black lines. Both lanes are from the same gel. Four yeast and two human libraries from mirRICH purified RNA were constructed. c, Library length distribution of bifurcated monosome (top) and disome (bottom) libraries by Agilent 2200 TapeStation. Libraries were bifurcated at the cDNA size selection step as shown in Extended Data Fig. 6b. d, Gene-level estimates for CDS occupancy, as in Fig. 1a, but here for mirRICH or Direct-Zol P1 nuclease RPF from the yeast lysate without HTS1 knockdown, purified by sucrose cushion. Both libraries relied on cDNA size selection rather than RNA size selection. The counts were deduplicated before analysis. e, Distribution of the log2 ratio of PCR deduplicated counts (corrected) versus uncorrected counts for each CDS from the libraries generated from the no HTS1 knockdown lysate, purified by sucrose cushion. The number of necessary PCR cycles used for library multiplexing is defined above. Two technical replicates per library condition were analyzed. f, Read length distribution represented as a fraction of mRNA mapping reads for the sucrose cushion purified mirRICH monosome and disome reads bifurcated by cDNA size-selected libraries from the HTS1 knockdown lysate. Two technical replicates per library condition were analyzed. Monosome replicates are in light blue and purple; disome replicates are in red and green. g, Read length distribution represented as a fraction of mRNA mapping reads for the sucrose cushion purified mirRICH monosome and disome reads bifurcated by cDNA size-selected libraries from the 293 T lysate. Two technical replicates per library condition were analyzed. Monosome replicates are in light blue and purple; disome replicates are in red and green. h, 5′ aligned ends and footprint length profile at initiating codons for P1 sub-disome (red line) and true disome (blue line) RPFs captured by mirRICH from human 293 T cell lysates. Material was purified by a sucrose cushion. The summed 5′ end profile is depicted at top and the contributions from each footprint length at each 5′ end are shown at bottom. Sub-disome and true disome-sized reads were analyzed separately.

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