Simultaneous generation of many RNA-seq libraries in a single reaction

Shishkin, A.A.; Giannoukos, Georgia; Küçükural, Alper; Ciulla, Dawn; Busby, Michele; Surka, Christine; Chen, Jenny; Bhattacharyya, Roby P.; Rudy, Robert F.; Patel, Molini M.; Novod, Nathaniel; Hung, Deborah T.; Gnirke, Andreas; Garber, Manuel; Guttman, Mitchell; Livny, Jonathan

doi:10.1038/nmeth.3313

Cited by 247 publications

(245 citation statements)

References 34 publications

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“…Extracted RNAs were then purified with RNA Clean & Concentrator™-5 (Zymo). After a dephosphorylation treatment, the RNA in each sample was ligated to a mixture of barcoded adapters in which each adapter had a unique barcode identifier according to our Massively Multiplexed RNA Sequencing method (25). After ligation, beads were rinsed with 1x PBS and detergents and then 5x PBS and detergents prior to pooling 3-4 IPs per new tube.…”

Section: Crosslinking and Immunoprecipitation (Clip) Analysismentioning

confidence: 99%

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

Chen

Blanco

Jackson

et al. 2016

Science

243

209

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Plunging into a domain of silence Female mammals have two X chromosomes. One must be silenced to “balance” gene dosage with male XY cells. The Xist long noncoding RNA coats the inactive X chromosome in female mammalian cells. Chen et al. show that the Xist RNA helps recruit the X chromosome to the internal rim of the cell nucleus, a region where gene expression is silenced. Xist is recruited to the domain through an interaction with the Lamin B receptor. This recruitment allows the Xist RNA to spread across the future inactive X chromosome, shutting down gene expression. Science , this issue p. 468

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Section: Crosslinking and Immunoprecipitation (Clip) Analysismentioning

confidence: 99%

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

Chen

Blanco

Jackson

et al. 2016

Science

243

209

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“…Cell lysis. The original RNAtag-seq protocol 10 was optimized for bacterial transcript analysis, and lysis methods involved enzymatic pretreatment and mechanical disruption, both of which can be deleterious to host RNA and inadequate for low yield samples. In this protocol, we have optimized the lysis of both host and bacterial cells either by using gentle bead disruption (for higher cell number; >10 6 ) or by enzymatic treatment (for lower cell numbers; <10 5 ).…”

Section: Comparison To Other Currently Available Methodsmentioning

confidence: 99%

“…Barcoded adapters were chosen from a set designed and vetted as part of the development of RNAtag-Seq 10 . These adapters were found to yield similar numbers of reads per sample and produce highly correlated transcriptional profiles when used to barcode replicate RNA samples derived both from bacteria and from mammals o Illumina Primer P5/P7 (12.5 µM) -see Table 2.…”

Section: Reagentsmentioning

confidence: 99%

“…Next, species-specific rRNA removal and reverse transcription enables ligation of a second adapter to the 3' end of the cDNA. The last step of PCR amplification then allows for strand-specific, quantitative sequencing and assembly of full-length transcripts in prokaryotic or eukaryotic species 10 . In order to adapt RNAtag-seq for simultaneous profiling of host and pathogen transcriptomes, the development of the following capabilities were required (Figure 1): firstly, the ability to effectively lyse both host and bacterial pathogen in a manner that allows recovery of RNA with high integrity and efficiency.…”

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confidence: 99%

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A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

et al. 2016

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EDITORIAL SUMMARY:This protocol enables simultaneous analysis of host and bacterial transcripts by RNA-Seq. Including procedures for efficient host and bacterial cell lysis, barcoding of samples, and analysis of both mammalian and microbial reads from mixed host-pathogen RNA-Seq data.TWEET: Simultaneous analysis of host and pathogen transcriptomes by RNA-Seq. Abstract The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. While RNA-Seq has greatly advanced our ability to study the transcriptomes of prokaryote and eukaryotes separately, limitations in existing protocols for generating and analyzing RNA-Seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-Seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low yield sample inputs and a computational pipeline to analyze both mammalian and microbial reads from mixed hostpathogen RNA-Seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach, suitable for large-scale studies, which will enable the field to obtain in-depth analysis of hostpathogen interactions in infection models. Introduction Intracellular bacterial pathogens, such as Mycobacterium tuberculosis, Salmonella enterica, Legionella pneumophilia, and Neisseria gonorrhea, spend a significant portion of their life-cycle surviving and replicating within host cells. The cellular interaction entails both a complex virulence program executed by the bacterial pathogen during infection 1 and activation of an orchestrated defense response by the host to counter the pathogen 2 . Genomic approaches have been employed in recent years to uncover substantial molecular details of this rich host-pathogen biology 3, 4 . However, technical constraints limit these studies to profiling either the host or the pathogen -while a comprehensive understanding of host-pathogen interactions requires simultaneous analysis of the associated gene expression changes in both the pathogen and the host 5 . The limitations of conventional protocols for simultaneous analysis of host and pathogen transcripts include 1) the inability to obtain high quality RNA with efficient lysis of both bacterial and mammalian host cells; 2) inefficient depletion of both microbial and mammalian ribosomal RNA species; 3) inability to simultaneously process polyadenylated and non-polyadenylated transcripts from low yield samples (>10ng of RNA); and 4) a lack of robust computational approaches for analyzing the often small subset of bacterial transcripts in infected cells or tissue. Recently, sever...

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“…Recent multiplexing approaches in RNA-seq such as RNA-tagSeq (Shishkin et al, 2015) and Ligation Mediated RNA sequencing (Hou et al, 2015) are set to further reduce costs of sequencing. Additionally, the number of sequenced genomes of plant hosts and their pathogens is rapidly increasing, facilitating dual RNA-sequencing studies [e.g.…”

Section: Next Generation Of Dual Approachesmentioning

confidence: 99%

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Naidoo¹,

Visser²,

Zwart³

et al. 2018

Current Issues in Molecular Biology

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RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

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Simultaneous generation of many RNA-seq libraries in a single reaction

Cited by 247 publications

References 34 publications

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

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