I have a complicated experimental system that is looking at changes in a region of the brain during learning. Past NGS has shown that, perhaps not surprisingly for brain tissue, there is a lot of animal to animal variability. Also - we cannot completely cleanly cut out the cells of interest, so the tissue is contaminated with other layers of the brain, other glial cells, etc which may or may not have the same gene expression affects. Other than doing LCM/single cell studies- I am looking for recommendations in the best way to plan the following experiment:
We have 6 time points. Planned to do 12 experimental animals and 6-12 control (will 6 be enough or do I need to do the same as experimental?)
Question1: Is it better to individually barcode 12 experimental animals and run on 2 lanes (to get ~30M reads per animal), or can I (perhaps to remove animal to animal variability, whch I am less interested in) pool animals, and do triplicate samples with 4 animals pooled in each? That way I would do 3 pooled experimental and 3 pool control animals. Would the triplicates give me enough statistical power?
Question2: Can I cross-use controls? So if I have the experimental animals undergoing learning for 24, 48, 72 hours etc, but the control animals are essentially identical animals grown in a cage without undergoing learning paradigms, can i make just 3 control animals for each time point and maybe pool them together as a general control group, or are batch effects expected?
Question 3: can I "virtually" pool? So for example, barcode each animal separately, but perhaps run them so each has 10M reads per animal and then pool identical number of reads per animal into 3 pools each of four animals with 30M reads per pool? What are the pros/cons/caveats of doing this?
Thanks!
We have 6 time points. Planned to do 12 experimental animals and 6-12 control (will 6 be enough or do I need to do the same as experimental?)
Question1: Is it better to individually barcode 12 experimental animals and run on 2 lanes (to get ~30M reads per animal), or can I (perhaps to remove animal to animal variability, whch I am less interested in) pool animals, and do triplicate samples with 4 animals pooled in each? That way I would do 3 pooled experimental and 3 pool control animals. Would the triplicates give me enough statistical power?
Question2: Can I cross-use controls? So if I have the experimental animals undergoing learning for 24, 48, 72 hours etc, but the control animals are essentially identical animals grown in a cage without undergoing learning paradigms, can i make just 3 control animals for each time point and maybe pool them together as a general control group, or are batch effects expected?
Question 3: can I "virtually" pool? So for example, barcode each animal separately, but perhaps run them so each has 10M reads per animal and then pool identical number of reads per animal into 3 pools each of four animals with 30M reads per pool? What are the pros/cons/caveats of doing this?
Thanks!
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