Atlas of zebrafish development at single cell resolutionsingle-cell RNA-seq technologies are rapidly increasing our understanding of organismal biology and provide new opportunities to understand the systems biology of development. We have initiated a project to map zebrafish development at single cell resolution and envision that this project will inform the entire community in their favorite tissue and gene of interest. The data utilized for generating our manuscript (Farnsworth DR, et al.; bioRxiv 2019) are hosted on this page and will be updated as new data are generated. All data will be integrated into ZFIN through a web portal that is currently in construction.
One of the most immediately useful and accessible pieces of data from the above manuscript is the supplemental tables that we use to annotate the clusters. Here they are in excel format:
We are currently working on providing the below files. We will soon have them deposited on the SRA and available here.
If you would like to perform your own analysis on the Atlas using the parameters we decided upon, the simplest method is to work from this *.rds file which can be read directly in R and analyzed using Seurat v2 (https://satijalab.org/seurat/install.html): [file soon]
Additionally, we have included the following .rmd markdown file that contains the commands we used to generate the *.rds during our analysis: [file soon]
If you would like to begin your own analysis, the matrices output from 10X Cellranger Count (v2.2.0, https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/count) are here as both individual replicates: [file] as well as the aggregated version of the six experiments we used in our recent publication: [file soon]
Lastly, the individual FASTQ files produced from sequencing are here: [file soon]
CRISPRscreenWe took advantage of CRISPR's high rate of mutagenesis to perform the first in vivo reverse genetic screen in a vertebrate. Our original paper in which we screen for electrical synapse defects can be found here: Nature Methods
We have developed a detailed protocol for the approach that can be found here: Methods in Cell Biology
And a critical improvement to the system can be found in this paper from the Mosimann lab. Our above protocol lays out how we have used Cas9 RNA and this is not the way to do such a screen these days. As Burger and colleagues show, using Cas9 protein/RNP complexes is much more efficient. Our ongoing screens in lab take advantage of Cas9 protein and we recommend it for and mutant screening.
One of the main goals in lab is to be able to quickly interrogate gene function in vivo in a vertebrate system. As zebrafish geneticists we love to be able to make mutations in genes and then assess the phenotypic outcome. However, classic forward genetic approaches, in which random mutations are generated in the genome, are notoriously slow at the stage of identifying the mutated gene. Its akin to finding a needle in a haystack. To speed the process we developed a next generation sequencing method to quickly map and identify mutated genes.See more here.