Abstract
RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible. Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq.
Highlights
Dupret et al (2018) modified this protocol to allow for genotyping of the individual samples
This involves removing a section of the larval tail and using this for genotyping, while the anterior part of the embryo is used for preparation of RNA
This is only possible at certain stages, and could potentially induce injury-dependent changes in gene expression. This procedure is labour-intensive and time-consuming, making it difficult to scale up. We have developed this protocol to enable us to do large-scale transcriptomics on individual zebrafish embryos (White et al, 2017)
Summary
Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq. The sample is DNase I treated to remove the genomic DNA, leaving RNA for the RNA-sequencing library making protocol. Adhesive plate seals (Thermo Fisher Scientific, catalog number: AB0558) 4. 5 mm stainless steel beads (Qiagen, catalog number: 69989) 5. Optional: TissueLyser Single-Bead Dispenser, 5 mm (Qiagen, catalog number: 69965) 6.
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