cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v3

Abstract

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .