A Revised Adaptation of the Smart-Seq2 Protocol for Single-Nematode RNA-Seq

Chang, Dennis; Serra, Lorrayne; Lu, Dihong; Mortazavi, A; Dillman, Adler R.

doi:10.1007/978-1-0716-0743-5_6

Cited by 11 publications

(13 citation statements)

References 26 publications

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“…In addition, the library construction method uses oligo-dT anchored primers for RNA capture, so it will not cause base imbalance in sequencing results due to GC content deviation. Previous studies have shown that ultra-low input RNA-seq analysis based on Smart-seq2 has been applied in animal liver, lymphoid cell, fungi, and single-nematode [ 35 – 38 ].…”

Section: Discussionmentioning

confidence: 99%

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

et al. 2021

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Background Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. Results Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. Conclusions The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

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Section: Discussionmentioning

confidence: 99%

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

et al. 2021

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“…To avoid potential amplification biases by different PCR cycles, we used 12 PCR cycles for cDNA amplification and eight PCR cycles for DNA (library) amplification in all samples. Together, this protocol contains four major modifications relative to the work of Macchietto et al (2017) and Chang et al (2021) : (1) We lysed worms through freeze-thawing; (2) we removed genomic DNA and performed this reaction in the same PCR tube as the cDNA synthesis reaction; (3) for the library preparation, we reduced the input of cDNA (to ∼1 ng); and (4) we only amplified the final library through eight cycles of PCR reaction. Raw reads of the 114 SWT samples were submitted to the European Nucleotide Archive (see Data access).…”

Section: Methodsmentioning

confidence: 99%

“…However, such methods are restricted to organisms that can be massively cultured and that stay synchronous throughout development. One alternative methodology is single worm transcriptomics (SWT), a method that has originally been introduced in two entomopathogenic nematodes of the genus Steinernema ( Macchietto et al 2017 ) and recently has been revised ( Chang et al 2021 ). Although such a method could in theory be adopted in many organisms, to the best of our knowledge, it has not yet been widely applied.…”

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confidence: 99%

Single worm transcriptomics identifies a developmental core network of oscillating genes with deep conservation across nematodes

2021

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High-resolution spatial and temporal maps of gene expression have facilitated a comprehensive understanding of animal development and evolution. In nematodes, the small body size represented a major challenge for such studies, but recent advancements have helped overcome this limitation. Here, we have implemented single worm transcriptomics (SWT) in the nematode model organism Pristionchus pacificus to provide a high-resolution map of the developmental transcriptome. We selected 38 time points from hatching of the J2 larvae to young adults to perform transcriptome analysis over 60 h of postembryonic development. A mean sequencing depth of 4.5 million read pairs allowed the detection of more than 23,135 (80%) of all genes. Nearly 3000 (10%) genes showed oscillatory expression with discrete expression levels, phases, and amplitudes. Gene age analysis revealed an overrepresentation of ancient gene classes among oscillating genes, and around one-third of them have 1:1 orthologs in C. elegans. One important gene family overrepresented among oscillating genes is collagens. Several of these collagen genes are regulated by the developmental switch gene eud-1, indicating a potential function in the regulation of mouth-form plasticity, a key developmental process in this facultative predatory nematode. Together, our analysis provides (1) an updated protocol for SWT in nematodes that is applicable to many microscopic species, (2) a 1- to 2-h high-resolution catalog of P. pacificus gene expression throughout postembryonic development, and (3) a comparative analysis of oscillatory gene expression between the two model organisms P. pacificus and C. elegans and associated evolutionary dynamics.

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“…In the single-worm RNA-seq method developed by Dillman and colleagues [ 14 , 15 ], a worm was cut, chemically lysed in a lysis buffer, and then subjected to reverse transcription using SMART-Seq2 reagents. Because the SMART reagents work best with isolated RNA as input [ 11 ] and the harsh lysis buffer used to chemically penetrate the worm’s thick cuticle could potentially degrade RNA molecules, we wanted to mechanically break worms, isolate RNA, and then use that RNA as input in our single-worm RNA-seq protocol.…”

Section: Resultsmentioning

confidence: 99%

“…Compared to standard RNA-seq that uses RNA from hundreds to thousands of worms as input, single-worm RNA-seq faces two major technical challenges: low input RNA from a single worm and low accessibility of that RNA due to the nematode’s thick surrounding cuticle. While the limited amount of input RNA can be overcome by adapting library preparation methods from single-cell RNA-seq, the harsh chemical lysis condition commonly used to penetrate the worm cuticle and access the RNA could potentially cause RNA degradation [ 12 – 15 ]. In the current study, we tested three different conditions for the mechanical lysis of worms and found an optimal condition to maintain RNA integrity.…”

Section: Introductionmentioning

confidence: 99%

Using single-worm RNA sequencing to study C. elegans responses to pathogen infection

et al. 2022

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Background Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. Results In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. Conclusions We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.

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A Revised Adaptation of the Smart-Seq2 Protocol for Single-Nematode RNA-Seq

Cited by 11 publications

References 26 publications

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

Single worm transcriptomics identifies a developmental core network of oscillating genes with deep conservation across nematodes

Using single-worm RNA sequencing to study C. elegans responses to pathogen infection

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