The Data

For this internship, gene expression data has been provided to us by our supervisor. Collecting this data followed several steps:

1. A Bacterial Artificial Chromosome was genetically engineered
2. Transgenic embryos were generated by injecting the BAC into zygotes
3. These embryos were cultivated to 30 hours post fertilization
4. The BAC attaches the gene for Green Fluorescent Protein to the regulatory apparatus for a gene whose expression is known to define the desired cell type (FoxA in this case). Therefore, when this gene -that is known to be highly expressed during the phase we are interested in- is expressed, GFP will also be expressed causing this particular cell type to fluoresce.
5. The Embryo is carefully disaggregated using both physical and chemical methods so that the gene expression of the cells is not affected.
6. Cells are run through flow cytometry to separate the GFP positive and negative populations (Archenteron cells vs All other cell types) as a method of Fluorescence Activated Cell Sorting (FACS).
7. The mRNA present in these cell populations is isolated, amplified (by rolling circle amplification), sequenced and quantified (Using Illumina RNA sequencing)
8. Normalization of the number of sequencing reads per gene is then performed according to the gene length and sum of all reads.
9. These sequencing reads are then mapped to the specific genomic loci.

The data we have is from two biological replicates (Samples from two distinct organisms which show biological variation), which is comprised of the normalized RNA count for both the GFP Positive and Negative populations for every gene in the transcriptome.

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