Sex-typing pure species embryos and the RNAseq experiment

Now that I have the gDNA, I've set up the PCR to test for sex. The PCR appears to have worked well. So far for P. campbelli (lanes 1-22) I have 10 males from 3 crosses and 12 females from 6 crosses. From P. sungorus (lanes 23-40) I have 10 males from 5 crosses and 8 females from 5 crosses. This should work well for the RNAseq experiment as I want 5 of each gender of each species. However, I will extract a few more P. campbelli males as it would be nice to have 5 litters instead of 3 that way every individual represents a different replicate cross.

Below is the gel. Positive bands mean that individual is a male, whereas negative bands indicate that the PCR failed and I am interpreting that to be a female. Alternatively failed reactions could be males and failed for some other reason - it's hard to tell the difference between these two alternatives. I am assuming that there will be some non-specific amplification in the females, so a smear indicates that the reaction would have worked had there been a Y chromosome (rather than failed outright) and is thus an actual female, whereas a completely blank lane indicates a likely failed reaction. I'll only use "females" who showed some non-specific binding rather than the "females" where there is no product in the well at all (fortunately, all the "females" have a smear, none failed completely).

As I have alluded to it numerous times, here is the outline of the experiment and the motivation for the sex-typing:

I want to compare the imprinting (and more generally, the entire expression profile) in Phodopus hybrids. As the hybrids show drastic parent-of-origin dependent growth differences, there should be some pretty striking parent-of-origin dependent differences in gene regulation (likely a species-specific breakdown of imprinting). The general approach is to compare the profile of the reciprocal hybrids with each other and then to expression of the parents. The between-hybrid comparison can tell me about imprinting and whether it is disrupted in any specific manner as well as whether there are any transcriptome-wide changes in gene expression. The hybrid-parent comparison can tell me about overall level of expression and if it is in fact different between hybrids, then I can use the parent species as a control for "normal" levels.

We are unsure of whether there will be sex-specific expression differences though and to account for that - there are genes that are predicted to show differences between the sexes during development - I will use 5 of each sex from each cross type. This results in 40 samples, 5 biological replicates per genotype, which will hopefully give me enough power to ask the questions I'm interested in.

The sex-typing from above will help me choose which samples will be the ones that I prepare.