These methods integrate next generation sequencing and flow cytometry imaging analysis. It can help answer interesting questions, like whether some phenotypic alterations are implicate, instead of the transcriptome, especially in the pollutant exposure environment. The main advantage of this simple method for us is that we can use it to measure the colocalization of biologically important protein, to interpret the outcomes of key differential gene expression at a functional level.
The implications of this technique extend toward the diagnosis of pathological and toxicological outcomes. As a normal imager approach, forsee has the ability to connect gene expression to protein function. Generally, individuals new to this method will struggle because of the high technical demands of the imaging and transcriptomic analysis.
To sequence the the human monocyte derived dendritic cell library, first load the sequencing and indexing reagents onto the sequencing biosynthesis and indexing reagent racks, respectively, and place the racks in a laboratory grade water bath for one hour, until all of the ice has melted, and the reagents are mixed thoroughly. While the reagents are thawing, power on the sequencer, connecting the computer to a network drive when the Do Not Eject drive appears, and launch the sequencer control software. Next, add about 100 milliliters of maintenance wash solution to each of the bottles in the sequencing biosynthesis reagent rack, and screw a funnel cap onto each bottle.
Add approximately 12 milliliters of maintenance wash solution to each 15 milliliter conical tube in the indexing reagent rack, and discard the caps. Then load both the racks onto the sequencer. Select Maintenance Wash within the sequencer control software, and follow the instructions for cleaning the sequencer fluid system.
Use a flashlight to visually inspect the flow cell for bubbles passing through the lanes, to make sure there is no leakage. When the wash sequence is finished, open the Sequence tab, and start a new run, directing the output data to a network drive. Upload a sample sheet for demultiplexing, and enter the appropriate reagent information.
Use a used flow cell to prime the system, with the sequencing reagents. Once the cluster generation is complete, remove the flow cell. And lightly spray the cell with water.
Wipe the flow cell dry with lens paper, followed by a light spray with 95%ethanol. After wiping the flow cell dry again, check the flow cell's surface against the light to make sure that it is clean, without debris or salt residue. When the prime step is finished, load the clustered flow cell, and begin the sequencing.
The sequence analysis viewer software will automatically be started. About 26 hours later, monitor the sequencing data quality via the high sequence control software, and sequencing analysis view software to assess the data quality, and for any troubleshooting. Then, when the sequence is finished, change the flow cell gasket, and perform a maintenance wash before beginning the next run.
After flow cytometric imaging of the pollutant-exposed dendritic cells, open ImageJ Fiji, and merge the 100 saved cell images for the non-exposed and pollutant-exposed human dendritic cell sample groups into individual files according to the treatment group. To analyze CD1d, and Lamp1 colocalization within the two populations, open the pollutant-exposed 100 merged cell image, and select Image, Color, and Split Channels to split the image into two individual images with a single fluorophore per channel. To create a scatter plot of the colocalized pixels, select Analyze, Colocalization, and Colocalization Threshold, and press Print Screen to save the scatter plot.
To calculate the Mander's colocalization coefficient for each single-cellular image, use the oval selection tool to select a single-cell image on the image file with the split channels, and select Analyze, Colocalization, and Colocalization Threshold again. Then select Channel 1 from the dialogue box for the region of interest, and click OK.When the coefficient has been calculated for all 100 cell images, import the results into an appropriate spreadsheet, and repeat the analysis for the control non-exposed cell sample. Using RNA sequencing and transcriptomic data analyses, several major altered gene clusters, including lipid metabolism and endocytic functions were identified in the pollutant-exposed DCs.
At the individual cell image level, HLADR positive CD11c positive dendritic cells can be gated, according to their CD1d and Lamp1 coexpression. Control non-exposed dendritic cells demonstrate minimal CD1d and Lamp1 protein colocalization, indicating a basal level of CD1d endocytic trafficking, and a high level of surface expression under physiological conditions. Whereas CD1d is retained in the late endocytic compartments of pollutant-exposed dendritic cells, confirming altered endocytic gene profiles in this experimental cell population.
After merging the individual cellular images into a single image file, the individual images can be split according to their fluorophore expression, and the colocalization of the pixel intensity between the two channels can be visualized in a scatter plot. The degree of colocalization between the CD1d and Lamp1 proteins in the two treatment groups can then be quantified using Mander's coefficients. If the protein intensity is hetergeneous between the colocalized and non-colocalized areas, the colocalized intensity will not be parallel to the colocalized areas, therefore it is also important to further assess the colocalization of the two proteins, based on the pixel intensity.
Once mastered, the image analysis can be completed in two hours, and the RNA sequencing setup can be completed in three hours, if each technique is performed properly. While attempting this procedure, it is important to remember to make sure that all the reagents are properly loaded in the correct positions, and that there's no leakage in the sequencer. Following this procedure, other methods, like micro RNA, exome, or mast cell sequencing can be performed to answer additional questions about global micro RNA expression, gene mutation, or DNA methylation, respectively.
So after it's development, this technique will help researchers in the field of cellular imaging analysis, and next generation sequencing to explore the effect of the environmental pollutant on gene expression and protein function. After watching this video, you should have a good understanding of how to set up the next generation sequencing, and the imager analysis of protein colocalization. Don't forget that working with a toxic pollutant chemicals in human blood samples can be extremely hazardous.
Precautions such as using a chemical fume hood, and personal protective equipment, should always be taken when performing these procedures.
