a, Schematic overview comparing three different library preparation methods for studying the transcriptome using nanopore sequencing. RMX, RNA adapter mix (provided with the SQK-RNA002 dRNA-seq library preparation kit); AMX, adapter mix (provided with the SQK-DCS109 dcDNA-seq library preparation kit). b, Nano3P-seq captures a wide range of RNA biotypes in a mouse brain nuclear/mitochondrial RNA sample. c, Integrative Genome Viewer (IGV) snapshot of reads generated with Nano3P-seq, mapped to the Ubb gene, illustrating the diversity of polyA tail lengths captured across different reads. The polyA tail region is shown in green. kbp, kilobase pairs; scaRNA, small Cajal body-specific RNA. d, Scatter plot of log transformed concentrations (amol µl⁻¹) and read counts of sequin genes (Pearson’s R = 0.93, slope = 0.93). Each dot represents a sequin. See also Extended Data Fig. 1h,i.

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a, Schematic overview of the transcriptional change that occurs during the MZT in zebrafish. b, Scatter plots depicting the correlation of mRNA log transformed RPM between biological replicates at three different time points during the MZT. c, Changes in mRNA abundance during the MZT (t = 2, 4, and 6 h.p.f.), relative to 2 h.p.f. Genes previously reported to have a ‘maternal decay mode’ are depicted in red. d, Bar plots depicting the abundance of different RNA biotypes captured by Nano3P-seq during the MZT (2, 4, and 6 h.p.f., shown in blue, green, and red, respectively). Statistical analyses were performed using the Kruskal–Wallis test. n = 3 biological replicates, data are presented as mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, P ≤ 0.001, ****P ≤ 0.0001. n.s., not significant (P > 0.05). e, Relative proportion of coding and non-coding RNAs captured using dRNA-seq (on polyA-selected samples), Nano3P-seq (on polyA-selected samples), and Nano3P-seq (on ribodepleted samples). f, Percentage of reads mapping to distinct biotypes captured using Nano3P-seq (on ribodepleted samples) (green), Nano3P-seq (on polyA-selected samples) (blue), and dRNA-seq (on polyA-selected samples) (light brown).

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a, PolyA tail length estimates of non-polyadenylated (curlcake 1) and polyadenylated (curlcake 2) synthetic RNAs sequenced with Nano3P-seq. See also Extended Data Fig. 1a–c. nt, nucleotides. b, Schematic overview of the standards used to assess the tail length estimation accuracy of Nano3P-seq. c, Box plots depicting tail length estimations of RNA and cDNA standards sequenced with Nano3P-seq. Values on box plots indicate the median polyA tail length estimation for each standard. d, PolyA tail length distribution of yeast, zebrafish, and mouse mRNAs represented as single-transcript values (left) and per-gene medians (right). e, PolyA tail length estimates across different RNA biotypes from mouse brain total RNA enriched in nuclear/mitochondrial RNA. Each dot represents a read. f, Replicability of median per-gene polyA tail length estimations of zebrafish embryonic mRNAs between two biological replicates for three different time points (2, 4, and 6 h.p.f.). g, Median per-gene polyA tail length distribution of zebrafish embryonic mRNAs across zebrafish developmental stages (2, 4, and 6 hpf, shown in blue, green, and red, respectively) in three biological replicates (shown as full lines, dashed lines, and dotted/dashed lines, respectively). h, Comparative analysis of mRNA abundances (shown as log₁₀(RPM) counts) of zebrafish mRNAs binned according to their annotated decay mode (maternal decay, zygotic activation-dependent decay, miR-430-dependent decay, and no decay) during early embryogenesis (t = 2, 4, and 6 h.p.f.). i, Median per-gene polyA tail length estimations of zebrafish mRNAs binned according to their decay mode (maternal, miR-430, zygotic, and no decay) at 2, 4, and 6 h.p.f. For Fig. 3h,i; statistical analyses were performed using the Kruskal–Wallis test. c,e,h,i, The number of observations included in the analysis is shown below each box and violin plot. Box plot limits are defined by lower (bottom) and upper (top) quartiles. The bar indicates the median, and whiskers indicate ±1.5× interquartile range.

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a, Comparison of polyA tail length distributions of reads mapping to khdrbs1a, illustrated at the per-gene level, measured at three time points during the zebrafish MZT. Annotations of the gene and two main isoforms are shown at the top of the panels, along with an IGV coverage track of the reads mapping to the gene. b,c, Comparison of polyA tail length distributions of reads mapping to two distinct isoforms (full and dashed outline) of elavl1 measured at three time points during the zebrafish MZT. Annotations of the gene and two main isoforms are shown at the top of the panels. a–c, Only isoforms with more than ten reads are shown. The number of reads included in the analysis is shown below each violin plot. P values have been computed using the Kruskal–Wallis test and corrected for multiple testing using the Benjamini–Hochberg method. Box plot limits are defined by lower (bottom) and upper (top) quartiles. The bar indicates the median, and whiskers indicate ±1.5× interquartile range. d, IGV coverage tracks of reads mapping to mouse processed small subunit rRNA (top track) and precursor SSU rRNA (bottom track), including a magnified image at the position known to be modified with m¹acp³Ψ (left). Reads mapping to SSU rRNAs were assigned to either ‘precursor’ or ‘processed’ isoforms on the basis of the overlap between 3′ end of the read and annotated end of the isoforms. Only reads with 3′ ends within ±10 nucleotides of the annotated end of an isoform were kept. Positions with a mismatch frequency lower than 0.1 are shown in gray. Middle, the mismatch frequency values in mouse precursor and processed SSU rRNA at the position known to be modified with m¹acp³Ψ (n = 2 biological replicates) are shown. Right, the per-site mismatch frequencies observed in reads mapping to mouse precursor SSU rRNA and mouse processed SSU rRNA are compared, showing that the only outlier is m¹acp³Ψ.

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a, Schematic overview of the standards used to assess the ability of Nano3P-seq to accurately quantify the base content of polyA tails. b, IGV snapshots of nucleotide composition in cDNA standard tails sequenced using Nano3P-seq. Gray regions indicate the mapped part of the reads, whereas colored letters indicate soft-clipped bases (unmapped), which are the base-called tails, after trimming the adapter. c, Probability of base composition (A, green; G, orange; C, blue; U, red) per position in the last 20 nucleotides of the cDNA standard tails. See also Supplementary Note 1. d, Percentage of reads belonging to groups classified on the basis of their polyA tail base composition. Some sequence examples belonging to different groups are illustrated below the bar plots. Samples in this analysis are embryonic mRNAs across zebrafish developmental stages (2, 4, and 6 h.p.f., shown in blue, green, and red, respectively) in three biological replicates and a control that includes sequin R1 and R2 groups of RNAs (gray). Statistical comparison of means was performed using the Kruskal–Wallis test. n = 3 biological replicates, data are presented as mean ± s.e.m. e, PolyA tail length estimation distributions of mRNA reads belonging to groups classified on the basis of their polyA tail base composition across zebrafish development stages (2, 4, and 6 h.p.f.). f, Left, IGV snapshots of reads mapping to zebrafish actb mRNA. Right, zoomed images of individual reads with different terminal bases (top, all-A reads; middle, Term-G reads; bottom, Term-U reads) are shown.

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