RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

The overall goal of this procedure is to assess expression levels of all genes and samples from Electroporated chick embryonic spinal cords. This is accomplished by first in ovo, poring the right side of early embryonic neural tubes and re incubating embryos to the desired stage. The second step of the procedure is to dissect successfully transfected neural tube halves in an RNA free environment and proceed to lysis as quickly as possible.

The third step is to isolate RNA and subject the samples to library preparation and high throughput sequencing. The final step is to process the data sets to remove sequencing artifacts. Map breeds to the genome and assign expression levels to each gene.

Ultimately, results can show differentially expressed genes through comparison of expression levels in control and experimental conditions. Visual demonstration of this method is critical as a neuro tube dissection. Steps are difficult to learn because we require familiarity with embryo manipulation.

For this demonstration, the embryos have been transformed with the GFP expressing construct at the neural tube. Prepare to spend two hours working on up to 10 embryos to collect one to two micrograms of total RNA per embryo. Under RNAs, free conditions, harvest the embryos using fine dissecting scissors and a SVE collection spoon transfer each embryo to cold.

DEPC treated. PBS used a solution for all of the neural tube isolation steps proceed by using fine tweezers to remove the entire flank region of an embryo between the limb buds. Move this section to fresh cold solution with the tissue dorsal side up.

Use two fine glass needles to perforate three spots on each dorsal lateral side of the embryo between the neural tube and parial mesoderm. Now introduce the two glass needles into the central perforation and move them apart along the AP axis. Do this through each perforation as needed to ultimately detach the paral mesoderm, remove and discard this tissue.

Next, turn the embryo sideways and introduce the tips of two tweezers between the neural tube and the nodal cord. Slide them apart along the AP axis to divide the structures. Finish removing larger pieces of meder tissue from the neural tube by gently securing it with tweezers while tweezing off the excess mesoderm.

Then gently transfer the neural tube to fresh cold solution. Using a glass pipette with a manual pump, collect all the needed neural tubes and keep them on ice before proceeding. Now, divide each of the neural tubes into two halves.

Insert sharp tip tweezers into the lumen and tear through the roof plate. Then use the tweezers to cut through the floor plate and divide the tissue in half. Using a fluorescent dissecting microscope, sort the electroporated from non electroporated halves.

Transfer the sorted neural tube halves to a 1.5 milliliter tube with fresh solution. Flick these tubes to get all the tissue down to the bottom. Next, spin down the tubes at 100 GS for a few seconds.

Then very carefully under bright lighting, aspirate the snat. If tissue is drawn up, slowly eject it and repeat the spin. Once the neural tube halves have been cleared of solution, add 70 microliters per neural tube, half of lysis buffer to the tube.

Flick the tube and incubate it at room temperature for 15 minutes every five minutes, vortex the tube vigorously and proceed with collecting the RNA to purify RNA For sequencing, have at least eight neural tube halves per sample. Other collections of lyce neural tubes stored at negative 80 degrees Celsius can be pooled into the collection. Start with thawing them on ice for 15 to 30 minutes, and then complete the thawing at room temperature.

Once thawed, vortex all the samples vigorously and let them sit for five minutes. Then vortex the samples again and centrifuge them at 15, 000 GS for one minute. To get rid of insoluble material, transfer the supernatant to a new tube and proceed with the extraction.

Using an RNA isolation kit, complete the process by eluding the RNA at the greatest possible concentration. This protocol requires a Galaxy website account to ensure the needed space quota. First, FTP upload the FASTQ files to the site unless quality scores are already encoded in Shred plus 33.

First, prepare the uploaded data sets using the FASTQ groomer tool under the NGS QC and Manipulation menu. Otherwise, under attributes, change the file type to FASTQ sender. Next, under the NGS QC and Manipulation menu, use the FAST QC tool to analyze the general quality of the data sets.

Select the FASTQ quality trimmer under the same menu to discard the three prime ends that are of low quality. A quality score of 20 or above should be processed. Now go to the clip tool under the same menu.

Set this tool to discard reads with less than 45 base pairs. Input the adapter sequence. Disable the option for discarding reads with unknown bases, and choose the output to include clipped and non clipped sequences.

Proceed with the trim sequences tool. Use it to trim the first bases of the read to remove the five prime sequence bias that is detected by FAST qc. The next step is to download and unpack the latest chicken genome assembly from the Illumina I genome site.

Then upload the whole genome faster format file and the gene's annotation GTF format file to the Galaxy History. Now map the reads for each sample to the genome by accessing the N-G-S-R-N-A analysis menu and selecting the top hat two tool. Select the uploaded fasta file from the Galaxy History as the reference genome.

Next, choose the cuff links tool under the same menu to build a predicted transcriptome. For each sample, choose the GTF file to be used as a reference annotation guide. Select Perform bias correction using the FASTA file for the sequence data and enable the use multi read correction option.

Then merge all the assemble transcriptomes using the cuff merge tool. With this tool, choose the GTF file for the reference annotation and the FASTA file for the sequence data. Now compare the BAM files generated by top hat two for each sample using the cuff TIFF tool still under the RNA analysis menu.

In the transcripts field, select the merge transcriptome generated by cuff merge and enable the options. Use multi read correct and perform bias correction using the FASTA file for the sequence data. The next step is to sort the differential tables Using the Q value column, select the sort tool from the filter and sort menu.

The sequences with significant difference between experimental conditions will have the lowest Q values on column 13. Now have a look at the coverage graphs. First, convert the BAM files into bed graph files using the conversion tool under the bed tools menu under the options, disable reporting of regions without coverage and choose to treat spliced entries as distinct intervals.

Also scale the coverage by a factor that normalizes all the coverages in the experiment. Then convert the resulting bed graph file to the big wig format using the tool in the convert formats menu and upload the big wig file as a custom track to the UCSE server. On the USCS genome browser page, use GAL four assembly and gene searches for visual coverage of the data sets using the described methods neural tubes expressing empty PS vectors were harvested for their RNA.

This was done in duplicate and the resulting RNA was a very high quality with an optimal RIN value of 10 as expected. Global comparison of the transcriptomes of the duplicated empty vector controls showed no clear difference and the Pearson correlation coefficient was point 99. Also, there was a clear increase in the number of reads mapped to the scratch two gene sequence in an experimental sample expressing the scratch two zinc finger domains.

Thus, the methods were sensitive enough to detect the predicted enrichment. After watching this video, you should be able to isolate the embryonic neuro tube and have a good understanding of how to process and compare IC dataset to find differentially expressed genes by following the silicon analysis steps in section four of the protocol.