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. 2020 May 22;11:2589. doi: 10.1038/s41467-020-16390-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Pie chart of TIF-cluster categories proportions in hen2-2 mutants. b Log₂ fold-change of TIF-cluster categories in hen2-2 compared to wild type. Colour code as indicated in a. c Scatterplot of TIF-cluster TSS/PAS pairs in hen2-2. Red arrow indicates TSS/PAS pairs within initiation regions. The colour scale represents 2D position enrichment density of clusters from low (grey) to high (yellow). d TIF-cluster sizes in wild type (n = 41,190) and hen2-2 (n = 48,225). Black arrow indicates enrichment of short TUs in hen2-2. e Distribution of short TIF-clusters sizes (<350 nt). sppRNAs have a median length of 93 nt. f The distribution of nascent gene body RNAPII transcriptional activity for genes with sppRNAs compared to all other genes from pNET-seq (8WG16) in wild-type Arabidopsis. For both classes tested: n = 1639. Gene body is defined as +200 nts from annotated TSS and −200 nts from annotated PAS. The boxes in d–f bounds the iQR divided by the median, and whiskers extend to a maximum of 1.5× iQR beyond the box. g Genome browser screenshot of full-length MPK20 mRNA and sppRNAs revealed in the hen2-2 track. h RT-qPCR for MPK20 sppRNA levels in wild type and hen2-2. With reverse transcriptase (+RT) and without (−RT). Data are presented as mean ± SEM from three independent experiments. Black dots indicate individual data points (n = 3). p-value between wild type and hen2-2 are derived from two-sided Student’s t-test. i Profiling of MPK20 sppRNAs by agarose gel northern blotting in wild type and hen2-2. UBI is a loading control. Blot picture is representative of one out of three biological replicates. TIF-clusters in hen2-2 and positions of northern probes for sppRNA (5′ probe) and control probe for mRNA (3′ Probe) are indicated. j PAGE northern blot in wild type and hen2-2. Loading control is the U6 snRNA. The blot picture is representative of one out of three biological replicates. Enrichment of sppRNA is indicated in i–j by a black line. Source data of a, b, h and i, j are provided in the Source Data file.