Total Nucleic Acid Extraction from Single Zebrafish Embryos for Genotyping and RNA-seq

[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.

Please cite this article as: . Total Nucleic Acid Extraction from Single Zebrafish Embryos for Genotyping and RNA-seq. Bio-protocol 12(01): e4284. DOI: 10. Individual embryos are collected into 96-well blocks. Embryos are lysed and total nucleic acid is bound to paramagnetic beads (SPRI). After washing, the nucleic acid is eluted from the beads using water ( Figure 1).

Figure 1. Schematic of the extraction workflow.
An overview of the different parts of the extraction procedure. The steps for each part are shown inside the coloured arrows.
Note: A clean working environment is essential. Use clean gloves and spray the bench, equipment, and gloves with RnaseZap, to avoid Rnase contamination.
1. Collect single embryos in each well of a 2 mL round bottom assay block. Collect the embryos in 9 μL of egg water using truncated pipette tips. Pre-chill the plate on dry ice, and lower the pipette tip to the bottom of each well, to freeze the embryo instantly. Label the plate on the front and side with the well numbers that contain collected embryos and the experiment name. Seal the plate using adhesive plate seals. Pull the seal on the sides and secure the plate. Do not pull 5 www.bio-protocol.org/e4284 them over the labelled side as the ink may come off when the seal is removed. Immediately place on dry ice for ~5 min. From this step, you can either proceed to extraction straight away or store the plate at -80°C until needed.
On the day of extraction 2. Remove Agencourt RNAClean XP/AMPure XP beads from the fridge to come to room temperature over 30 min. Shake well to resuspend beads.
3. Prepare 70% ethanol fresh. You will need ~700 μL per well. 7. Remove the plate from the dry ice and allow time to defrost. Dry the top of the plate with a clean tissue and seal using adhesive foil plate seals. Do not push the seal inside the wells, as this will make it more likely that the bead will pierce the seal in the TissueLyser.
Alternatively, if using a heat sealer, seal the plate 6 times using a heat seal at 170°C for 3 s. adapter is completely flat and the other side is not. Make sure that the flat side of the adapter is on top of the plate and that the adapter is tight. We recommend testing the integrity of your plate seals on a plate containing water before processing real samples.
8. Install Plate Adaptor Set onto TissueLyser II, according to manufacturer's instructions. Arrange 2 mL assay blocks on the adaptor. Make sure the adaptor is balanced, that the plates are aligned properly between the adaptors, and the adaptor is not at an angle. Run protocol at 12 Hz frequency for 5 min at room temperature. 9. When the Tissue Lyser program ends, briefly spin the sample plate to collect the lysate at the bottom of the plate. If the lysate does not appear homogeneous, rerun the program at 12 Hz at two minutes increments until the lysate appears homogenous.
Note: The following steps need to be done under the fume hood. 17. In the final wash, thoroughly remove any residual ethanol using a multichannel pipette without disturbing the beads.
Note: You can now leave the fume hood.
18. Air dry sample at room temperature for 10-15 min, or until there is no remaining trace of liquid and the beads appear dry. Do not over dry, or else re-suspending the beads becomes difficult. 19. Add 58 μL of RNase-free water and re-suspend beads using pipette. DO NOT VORTEX. Using the same tip, gently flush the beads down the tube by pipetting the water over the beads, until all the beads are in suspension.