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. 2022 Apr 5;12:5718. doi: 10.1038/s41598-022-09638-3

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© The Author(s) 2022

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Assessment of riboPLATE-seq IP specificity. (a) Depletion calculated per-sample as the log2-ratio of the sum of all spike-in or gene-aligned counts in the riboPLATE-seq library over the same sum in the sample’s paired PLATE-seq library, using DESeq2-normalized counts (median of ratios normalization, R v4.0.5), in Wi-38 cells. Spike-ins show more significant depletion than genes in almost all wells (mean spike-in RA 0.25; mean genomic RA 1.04; Wilcoxon signed-rank test p = 1 × 10^–9) (b) The same information in (a), presented as the per-well difference in depletion ratios for ERCC and the background genome, demonstrating significant depletion of spike-in RNA with IP in most libraries (mean log2 depletion ratio − 2.1). (c) Relationship between transcript abundance (in PLATE-seq) and RA (riboPLATE/PLATE-seq) for coding genes and non-coding RNA (ncRNA) in Wi-38 cells. ncRNA are heavily depleted (RA < 0) at all expression levels, to a greater extent than almost all genes. (d) Relationship between transcript abundance and RA for ERCC spike-ins vs coding genes in Wi-38, demonstrating a pronounced pattern of depletion in RA for spike-ins relative to mRNA across all expression levels. (e) Relationship between PLATE-seq transcript abundance and RA for coding genes vs ncRNA in TS-543 cells. (f) The corresponding plot to e derived from ribosome profiling and RNA sequencing data of TE and RNA-seq transcript abundance in TS-543 cells. Though the shape of the distribution is different, ncRNA still demonstrate lower TE than mRNA at higher expression levels.