Gene expression - measured as steady state RNA levels:
Did the salt stress experimental treatment change gene expression?
Which variety (Agami or M103) exhibited the most change?
Which tissue type (root or shoot) exhibited the most change?

Alternative splicing - measured as relative abundance of splice variants per gene:
Did the salt stress experimental treatment change splicing?
Which variety (Agami or M103) exhibited the most change in response to salt?
Which tissue type (root or shoot) exhibited the most change in response to salt?

Combining methylation and AS analyses - how to present it:

• Report genes that are differentially spliced between treatment and control
• Report genes that are differentially methylated between treatment and control
• Report the overlap between the above two sets
• Gene Ontology enrichment analysis of each of the above groups, followed by discussion of what the biological meaning might be

There are known genes or regions that are thought to be involved in salt tolerance in rice. Many of the papers examine whether these genes or regions appeared in their equivalent of the above sets. This is another aspect of the analysis we should do, following the lead of the other articles published in this area.

Abstract for Frontiers in Plant Science: Alternative splicing is the formation of multiple transcript isoforms from a single gene, depending on environmental or intracellular factors. In plants, exposure to abiotic stress triggers genome-wide changes in splicing patterns, enriching for stress-specific transcript isoforms. In rice, abiotic stresses, such as excess salt exposure, can also alter DNA methylation, an epigenetic modification that can enhance stress tolerance. A connection between DNA methylation and gene expression has been established, however, little is known about the role of DNA methylation on differential splicing. Therefore, we investigated the role of methylation in splice site selection during salt stress in rice. We utilized RNA-Seq and whole genome bisulfite sequencing on shoot and root tissue of the M103 and Agami rice cultivars exposed to either control or salt treatments. Our results showed varying gene expression, methylation, and splicing due to salt treatment, tissue, and cultivar. By analyzing methylation and splicing together, our results showed a correlation between methylation and splice site choice.

COMPARISONS
Agami shoot salt vs M103 shoot salt
Agami root salt vs M103 root salt

Agami shoot salt vs Agami shoot control
Agami root salt vs Agami root control
M103 shoot salt vs M103 shoot control
M103 root salt vs M103 root control

GENERAL QUESTIONS
What genes are differentially expressed?
What GO terms are enriched in the differentially expressed genes?

What genes are differentially spliced?
What GO terms are enriched in the differentially spliced genes?

What genes are differentially methylated?
What GO terms are enriched in the differentially methylated genes?

What is the overlap of differentially methylated genes and differentially expressed genes?
What is the overlap of differentially methylated genes and differentially spliced genes?

SPECIFIC QUESTIONS
Estimate percentage of methylated cytosines to total cytosines across CG/CHG/CHH contexts for each tissue and variety.
Estimate DNA methylation density within body of protein coding genes and transposable elements across CG/CHG/CHH for each tissue and variety.
Examine known salt tolerance genes in rice for differential methylation, expression, and splicing.
Examine principal component analysis comparing gene expression across all samples to see if tissue, variety, or treatment had a major contribution in gene expression variation.

Additional questions will be needed to fully explore the relationship between alternative splicing and methylation. I will add additional questions and comparisons as needed.

Closing ticket for now.