Here we describe a rRNA depletion protocol based on RNase H digestion using antisense oligonucleotides (ASOs) specifically designed for C. elegans cytoplasmic rRNA (Fig. 1A). We suggest that this rRNA depletion protocol is applicable to RNA-seq applications where the yield of mRNA enrichment should be independent of poly(A) status, or any application which benefits from the removal of rRNA sequences, such ribosome profiling, or sequencing of non-coding RNAs other than rRNA

Ambros, Victor R.

Duan, Ye

Sun, Yongming

English

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University of Massachusetts Medical School eScholarship@UMMS University of Massachusetts Medical School Faculty Publications 2020-09-22 RNA-seq with RNase H-based ribosomal RNA depletion specifically designed for C. elegans Ye Duan University of Massachusetts Medical School Et al. Let us know how access to this document benefits you. Follow this and additional works at: https://escholarship.umassmed.edu/faculty_pubs  Part of the Bioinformatics Commons, Genetics and Genomics Commons, and the Molecular Biology Commons Repository Citation Duan Y, Sun Y, Ambros VR. (2020). RNA-seq with RNase H-based ribosomal RNA depletion specifically designed for C. elegans. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.17912/micropub.biology.000312. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1901 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in University of Massachusetts Medical School Faculty Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact Lisa.Palmer@umassmed.edu.  RNA-seq with RNase H-based ribosomal RNA depletion specificallydesigned for C. elegansYe Duan1, Yongming Sun2 and Victor Ambros1§1University of Massachusetts Medical School, Worcester, MA 01605, USA 2Integrated DNA Technologies, Inc., Redwood City, CA 94065, USA§To whom correspondence should be addressed: victor.ambros@umassmed.eduFigure 1: A. Protocol for depletion of ribosomal RNAs (rRNA) from samples of C. elegans RNA. Total RNA wasextracted from worm lysates, heat denatured to melt secondary structure, and hybridized to a molar excess of the DNAantisense oligo (ASO) mixture. rRNA annealed to their complementary oligonucleotides were digested by thermostableRNase H, followed by removal of genomic DNA and ASOs with DNase. The reaction was then passed through a sizeselection column to desalt the sample and remove the truncated rRNA fragments and small RNAs less than 150 nt (e.g.,tRNAs), and the RNA was cloned and sequenced. B. Alignments of ASOs to C. elegans 26S, 18S, 5.8S and 5S ribosomeRNAs (rRNA). Some ASOs have complementarity to both a 5’ segment and a 3’ segment of 5.8S sequence. Images areprimarily generated by SnapGene Viewer 5.1.4.1. C. Sequencing read coverage (linear scale; image generated by IGV2.7.2 (Robinson et al., 2011)) at the Y59C2A.2 and C50C3.2 mRNA loci, corresponding to the top two predicted off-targetsites of ASOs (red bar; see text for discussion). Note that the off-target depletion of mRNA sequence appears to be localand does not appreciably impact read density in other regions of those genes. Reads were from 4 libraries of differentgenotypes. D. Sequencing read coverage (linear scale) at the C. elegans primary rDNA locus from 4 libraries of differentgenotypes. We suggest that the reads flanking the rRNA come from primary rRNA transcripts, which were not targeted byASOs (Ellis et al., 1986). E. Biotype proportions of reads from 12 RNA-seq libraries of 4 different genotypes preparedbased on the rRNA depletion. F. Biotype proportions of reads in (E) after manually removal of signal recognition particle 9/22/2020 - Open Access (srpR) RNA reads, which are predominantly enriched. The lower panel shows the proportions of the cytoplasmic rRNAreads (with ASOs) and mitochondrial rRNA reads (without ASOs) among the rRNA reads.DescriptionRNA-seq is widely used for the quantitative analysis of transcriptomes in the context of studies of gene expression andregulation (Mortazavi et al., 2008; Ozsolak and Milos, 2011; Wang et al., 2009). Generally, RNA-seq protocols employpoly(A) selection for mRNA enrichment. However, poly(A) based enrichment is subject to potential bias depending on thepoly(A) status of various mRNAs, which could be particularly undesirable in the context of studying post-transcriptionalgene regulatory mechanisms, such as miRNA repression (Wu et al., 2006). Therefore, ribosomal RNA (rRNA) depletionis a desirable alternative strategy to enrich for mRNA sequences in RNA-seq sample preparation (Zhao et al., 2014).However, currently available rRNA depletion toolkits were designed for either mammals or bacteria, and hence do notoffer an efficient option for rRNA depletion of RNA samples from certain experimental organisms, such as C. elegans.Here we describe a rRNA depletion protocol based on RNase H digestion using antisense oligonucleotides (ASOs)specifically designed for C. elegans cytoplasmic rRNA (Fig. 1A). We suggest that this rRNA depletion protocol isapplicable to RNA-seq applications where the yield of mRNA enrichment should be independent of poly(A) status, or anyapplication which benefits from the removal of rRNA sequences, such ribosome profiling, or sequencing of non-codingRNAs other than rRNA.We designed 120mer DNA ASOs complementary to 26S, 18S and 5.8S rRNA sequences and 60mer ASOscomplementary to 5S rRNA (Fig. 1B). We used thermostable RNase H and optimized protocols at high temperature forthe ASO-rRNA duplex annealing and digestion to reduce the formation of secondary structure of rRNA and off-targetannealing of the ASOs. We found that among the 12 RNA-seq libraries (4 different C. elegans genotypes) prepared withour rRNA depletion protocol, the cytoplasmic rRNA (26S, 18S, 5.8S and 5S) reads only comprised < 0.2 % of the totalreads (Fig. 1E-F), indicating that our protocol is effective for removing rRNA. Highlighting the specificity of thedepletion, we observed reads from rRNA precursor transcripts (Fig. 1D) and mitochondrial rRNA (Fig. 1F, lower panel)due to the absence of ASOs against those sequences. As expected, the rRNA-depleted datasets contained a largerepresentation of other non-coding RNA sequences, further supporting that our protocol enriches for RNAs lackingpoly(A) (Fig. 1E-F). The performance of our depletion protocol appears to be similar to that of two independentlydeveloped similar protocols for C. elegans-specific rRNA depletion (Arribere et al., 2016; Barucci et al., 2020).Interestingly, we found that a large proportion of the non-coding RNA reads after RNA depletion correspond to the signalrecognition particle RNA (srpR) (Regalia et al., 2002). A similar (although somewhat less prominent) enrichment of srpRwas also observed in the libraries of Barucci et al., 2020 and Arribere et al., 2016. The srpR reads were manually removedin our analysis (Fig. 1F); however we suggest that later iterations of our protocol could include ASOs corresponding tosrpR, as well as mitochondrial rRNA and cellular primary rRNA transcripts.Although we did not directly compare the genome-wide distribution of mRNA reads from this ASO protocol to that ofmRNA reads from other approaches for mRNA enrichment, we did conduct a computational assessment of the potentialfor off-target effects of our ASO rRNA depletion. We adopted a conservative assumption that, for an ASO to triggerRNase H digestion of an mRNA sequence, it should match the mRNA with a melting temperature of no less than 20oCbelow the temperature at which the annealing and RNase H digestion were performed. Using BLAST, we identified onlytwo potential matches between the ASOs and the C. elegans transcriptome that met this criterion (Morgulis et al., 2008).Interestingly, these two sites do seem to locate in regions of their respective transcriptional units where reads wererelatively depleted locally, suggesting that the ASOs may have triggered off-target depletion at these sites (Fig. 1C).However, the apparent sequence depletion in these two instances was only restricted to regions around the ASO off-targetmatch sites, and the alignment distribution for each gene in sequences other than the off-target sites seems to be relativelyunaffected (Fig. 1C). From this analysis, we suggest that the ASO rRNA depletion method results in essentially negligibleoff-target mRNA depletion.MethodsRequest a detailed protocolrRNA depletion and RNA-seqHarvested worms were washed with M9 medium and flash-frozen in liquid nitrogen. The worm pellets were lysed byQIAzol (Qiagen, Cat: 79306) as previously described (McJunkin and Ambros, 2017). ASOs and total RNA were mixedwith approximate 2:1 molar ratio (the quantity of ASOs calculated assuming rRNAs comprise > 90% of total RNA).Higher molar ratios of up to 6.5:1 were tested but did not result in observable improvement in depletion efficacy.Accordingly, a mixture containing final concentrations of 0.1 mM of each ASO, 50 ng/µl total RNA, 10 mM Tris-HCl (pH= 8.0), 100 mM NaCl, 1 mM EDTA and 0.8 U/µl RNase Inhibitor (NEB, Cat:M0314) was incubated at 95 °C for 2 minfor denaturation and then 65 °C for 30 min for annealing. We have also tested annealing conditions with gradual decreasein temperature (0.5-2 °C per minute) but no apparent improvement in depletion efficacy was observed. To digest the 9/22/2020 - Open Access rRNA, thermostable RNase H and the buffer (MCLAB, Cat:HTRH-100) were preheated to 65 °C and added to thereaction (while maintaining the reaction at 65 °C) to obtain a final concentration of 0.2 U/µl RNase H and the reaction wasfurther incubated at 65 °C for 40 min, and then chilled on ice. Other digestion temperatures from 30 to 85 °C were alsotested, and 65 °C was determined to be optimal for reducing rRNA secondary structure without compromising overallRNA integrity of the samples. To digest the ASOs, Turbo DNase (Invitrogen, Cat:AM2238) and the buffer were added toa final concentration of 0.1 U/µl and the reaction was incubated at 37 °C for 25 min. mRNA was then purified by RNAClean & Concentrator-5 Kit (ZYMO, Cat:R1015) as described in (Zhang et al., 2012). The RNA-seq libraries wereconstructed by NEBNext Ultra II RNA Library Prep kit (NEB, Cat:E7775, E7335, E7500) and sequenced by IlluminaNextSeq 500 system.Data analysisAdaptor sequences were trimmed and reads shorter than 15 nt were filtered out from analysis by Cutadapt/1.4.1 (Martin,2011). For the analysis shown in Fig. 1F, the srpR reads were removed by initially mapping with Bowtie2/2.3.4.3(Langmead and Salzberg, 2012), and either total filtered reads (Fig. 1E) or the remaining reads after srpR removal (Fig.1F) were mapped to C. elegans genome (WBcel235) by Star/2.5.3 with default parameters (Dobin et al., 2013). Mappingdata was sorted and processed by Samtools/1.9 (Li et al., 2009). Gene counting was performed using featureCounts (Liaoet al., 2014).ReagentsThe ASO sequences in this research are available upon request.Acknowledgments: We thank Caifu Chen and Nicole Sponer at Integrated DNA Technologies, Inc. for help in developingthe rRNA depletion protocol. We thank Isana Veksler-Lublinsky from Ben-Gurion University of the Negev, Beer-Sheva,Israel for advice on data analysis.ReferencesArribere JA, Cenik ES, Jain N, Hess GT, Lee CH, Bassik MC, Fire AZ. 2016. Translation readthrough mitigation. Nature534: 719-23. PMID: 27281202.Barucci G, Cornes E, Singh M, Li B, Ugolini M, Samolygo A, Didier C, Dingli F, Loew D, Quarato P, Cecere G. 2020.Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants. Nat Cell Biol 22:235-245. PMID: 32015436.Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. 2013. STAR:ultrafast universal RNA-seq aligner. Bioinformatics 29: 15-21. PMID: 23104886.Ellis RE, Sulston JE, Coulson AR. 1986. The rDNA of C. elegans: sequence and structure. Nucleic Acids Res 14: 2345-64. PMID: 3960722.Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9: 357-9. PMID: 22388286.Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project DataProcessing Subgroup.. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078-9. PMID:19505943.Liao Y, Smyth GK, Shi W. 2014. featureCounts: an efficient general purpose program for assigning sequence reads togenomic features. Bioinformatics 30: 923-30. PMID: 24227677.Martin, M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10-12. DOI: https://doi.org/10.14806/ej.17.1.200McJunkin K, Ambros V. 2017. A microRNA family exerts maternal control on sex determination in C. elegans. GenesDev 31: 422-437. PMID: 28279983.Morgulis A, Coulouris G, Raytselis Y, Madden TL, Agarwala R, Schäffer AA. 2008. Database indexing for productionMegaBLAST searches. Bioinformatics 24: 1757-64. PMID: 18567917.Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomesby RNA-Seq. Nat Methods 5: 621-8. PMID: 18516045.Ozsolak F, Milos PM. 2011. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12: 87-98. PMID:21191423.Regalia M, Rosenblad MA, Samuelsson T. 2002. Prediction of signal recognition particle RNA genes. Nucleic Acids Res30: 3368-77. PMID: 12140321. 9/22/2020 - Open Access Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. 2011. Integrative genomicsviewer. Nat Biotechnol 29: 24-6. PMID: 21221095.Wang Z, Gerstein M, Snyder M. 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10: 57-63.PMID: 19015660.Wu L, Fan J, Belasco JG. 2006. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103: 4034-9.PMID: 16495412.Zhang Z, Theurkauf WE, Weng Z, Zamore PD. 2012. Strand-specific libraries for high throughput RNA sequencing(RNA-Seq) prepared without poly(A) selection. Silence 3: 9. PMID: 23273270.Zhao W, He X, Hoadley KA, Parker JS, Hayes DN, Perou CM. 2014. Comparison of RNA-Seq by poly (A) capture,ribosomal RNA depletion, and DNA microarray for expression profiling. BMC Genomics 15: 419. PMID: 24888378. Funding: NIH grants R01GM088365, R01GM034028, and R35GM131741 (VA)Author Contributions: Ye Duan: Conceptualization, Data curation, Methodology, Investigation, Writing - original draft,Writing - review and editing. Yongming Sun: Methodology. Victor Ambros: Conceptualization, Funding acquisition,Supervision, Writing - review and editing.Reviewed By: AnonymousHistory: Received September 11, 2020  Revision received September 20, 2020  Accepted September 21, 2020 Published September 22, 2020Copyright: © 2020 by the authors. This is an open-access article distributed under the terms of the Creative CommonsAttribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original author and source are credited.Citation: Duan, Y; Sun, Y; Ambros, V (2020). RNA-seq with RNase H-based ribosomal RNA depletion specificallydesigned for C. elegans. microPublication Biology. https://doi.org/10.17912/micropub.biology.000312 9/22/2020 - Open Access

RNA-seq with RNase H-based ribosomal RNA depletion specifically designed for C. elegans

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RNA-seq with RNase H-based ribosomal RNA depletion specifically designed for C. elegans

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