Extended Data Fig. 1: Fiber-seq-derived ACRs show expected patterns at ATAC ACRs and transcription start sites and expected correlation with expression.

A) Small molecule mass spectrometry data on nucleotides purified from various samples with known and unknown quantities of m6A and adenine (dA). Shown is the log-scale of the signal-to-noise ratio for each sample. Specifically, this includes plasmid DNA isolated from bacteria that lack any m6A-MTases. As positive controls these samples were treated with a non-specific m6A-MTase before nucleotide isolation. For maize, samples were prepared as for Fiber-seq with or without m6A-MTase-treatment. In addition, standards containing defined amounts of m6A and dA were used. The red dashed line corresponds to the signal observed in untreated maize genomic DNA, whereas the solid blue line shows essentially the limit of detection of m6A signal-to-noise for this assay based on the sample that has no m6A injected with a similar amount of total dA. (B) Schematic illustrating the calculation of the FIRE accessibility score, a measure of Fiber-seq-derived chromatin accessibility that allows direct comparisons to ATAC-seq-derived chromatin accessibility. Shown are screenshots of two FIRE ACRs with individual fibers showing FIRE elements (red) of different length, in addition to methyltransferase-sensitive patched in purple and non-methylated regions as grey lines. Black boxes mark the respective FIRE ACRs (black bars on top). For any given window, the FIRE accessibility score is calculated as the number of bases annotated as FIRE elements (red) divided by the total number of bases across all fibers mapping within this window (red, purple for methyltransferase-sensitive patches not annotated as FIRE elements, grey for not methylated). FIRE accessibility scores are shown for the two example ACRs. (C) Correlation between FIRE accessibility scores for Fiber-seq replicates 1 and 2. Each dot corresponds to ACRs where both replicates have >10x coverage. (D) Correlation of Tn5 insertions for union ACRs identified in ATAC-seq replicates 1 and 2. (E) m6A methylation peaked at the centre of ATAC-seq derived ACRs in paired samples. (F) m6A methylation rate peaked immediately upstream of CAGE-defined transcription start sites (TSSs), with phased nucleosomes apparent downstream of TSSs. Average strength of m6A methylation rate upstream of TSSs was monotonically related to expression level of respective downstream genes (expression deciles). The well-phased-nucleosome signal was strongest for highly expressed genes and faded for lowly expressed ones, as expected. (G) Methyltransferase-sensitive patches (MSPs) larger than 100 bp constituted the majority of the m6A signal at TSSs, while MSPs shorter than 100 bp showed patterns consistent with well-positioned nucleosomes. MSP scores were calculated in aggregate for each non-overlapping 20 bp window in the region 750 bp upstream and 1 kb downstream of each TSS (see Methods). (H) Aggregate plot of Tn5 insertions/base in the 1 kb window upstream of TSSs stratified by downstream gene expression for paired ATAC-seq data, comparable to (F). (FIRE elements supporting FIRE ACRs are in red, purple indicates methylation sensitive patches (see Fig. 1). (I) Aggregate plot of FIRE ACRs stratified into ten deciles based on their FIRE accessibility score. For each FIRE score accessibility decile, the number of Tn5 insertions at each bp within 2 kb of the FIRE ACR centre is shown. Accessibility measured by ATAC-seq and Fiber-seq is monotonically correlated. Highly accessible FIRE ACRs tend to show neighbouring FIRE ACRs (symmetric signal at highest decile). This signal is in part due to FIRE ACRs in low-mappability LTR retrotransposons (see Fig. 2). (J) The single-molecule method Fiber-seq outperforms single-cell ATAC-seq as a quantitative measure of chromatin accessibility. 39,132 ACRs were identified as shared FIRE ACRs in dark-grown maize leaves and ATAC ACRs in a pseudobulked leaf sample (GSM4696890) from Marand et al. The percentage of cells containing at least one Tn5 insertion within a shared ACR (% cells accessible) is compared to the percentage of actuated fibers (that is, with a called FIRE element, % actuated Fibers within a given ATAC ACR) underlying the same shared ACR. Each dot represents one shared ACR. Hexbin color reflects the number of dots.