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Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq   

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Original research article

A brief version of this protocol appeared in:
PLOS Pathogens
Apr 2017

Abstract

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds.

Keywords: RNA-seq, Transcriptome, C. elegans, S. carpocapsae

Background

Low input RNA-seq protocols and amplification kits, such as Smart-seq (Takara Bio, USA, Inc) and SuperAmp (Miltenyl Biotec, Inc), have been increasingly developed and commercialized as a response to the growing prevalence of low input RNA-seq studies based on small tissues, single microorganisms, and single cells. These studies often explore and address heterogeneous gene expression among individuals of a certain population, such as a population of cells, a complex tissue, or a population of microscopic organisms. Improvements and adaptations of low input RNA-seq protocols for microscopic organisms, such as nematodes, will greatly benefit the field of nematology by allowing for the analysis of gene expression heterogeneity at the single nematode level. Here we have adapted the single cell RNA-seq protocol, Smart-seq2 (Picelli et al., 2013 and 2014; Trombetta et al., 2014), for single nematode RNA-sequencing. We successfully utilized adapted versions of this protocol in the transcriptomic analysis of the insect-parasitic nematode, Steinernema carpocapsae (Lu et al., 2017) as well as in the analysis of individual embryos and L1 larvae from two Steinernema and two Caenorhabditis species including C. elegans (Macchietto et al., 2017), but this protocol can be adapted for any species of nematode. While this protocol will work on nematodes without already sequenced genomes or transcriptomes, we limit our computational analysis to organisms with published genome annotations, such as S. carpocapsae (Dillman et al., 2015). Our need for single nematode RNA-sequencing arose as a method to circumvent the limitations of working with samples with low-inputs of RNA. For example, many of our in vivo experiments limited the number of nematodes we could utilize. Single nematode RNA-seq has allowed us to efficiently obtain high resolution gene expression data from these nematodes. The protocol has also enabled us to collect individual embryos to map out time courses of nematode embryonic development for comparative transcriptomics across multiple species. The development and advancement of low input RNA-seq protocols will aid investigators in circumventing issues related to using individual organisms and specialized/limited samples.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Serra, L., Chang, D., Macchietto, M., Williams, K., Murad, R., Lu, D., Dillman, A. R. and Mortazavi, A. (2018). Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq. Bio-protocol 8(4): e2729. DOI: 10.21769/BioProtoc.2729.
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