Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-5-17
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Test Nuclei Loss
Background
- We've noticed that we are getting ~75% loss of nuclei with every centrifugation during our adapter ligation.
- Here are some notes from other lab members about that loss:
- Brandon - Amount of loss was about how much he gets from working with nuclei
- Blue - normally gets only ~25% loss of nuclei with every spin. Suggested that 6000xg speed centrifugation was too high and may be popping the nuclei. However, stickiness of nuclei was something that we could not really change with centrifugation. Also could try PBSe or 2% BSA that might help a bit
- Today, we'll be testing whether we are centrifuging too quickly, causing major sample loss. Yesterday, Huy tried the experiment from 900xg to 6000xg and noticed that the pellet became too hard to break up >3000xg.
- Yesterday, we had some difficulty determining how to count the nuclei. Initially, we gated the automated hemocytometer to 4um to 7um. However, Blue said he counts everything >4um. The reason why is because 4um can be potentially just debris and the smaller peak at 10um may either be doublets or even variability in nuclei size.
- Consequently, today, we need to figure out how to more accurately count the nuclei. One potential method would be to use the DNA content of the nuclei. This potentially may be less accurate than cell counts, but it will help us at least move past the problem of
Procedure
- Below are the speed conditions we want to test for this experiment:
900xg 15min 1500xg