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Old 07-22-2015, 04:22 AM   #1
F.L.
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Location: luxembourg

Join Date: Jul 2015
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Default Methyl-Seq: Lib prep (MspI;HpaII) for mouse samples causes issues for human samples

Hello,

I have a question concerning whole genome enzymatic digestion in preparation for NGS. Currently, we digest mouse tissue and cell line samples, using the standard enzymes MspI and HpaII, as an intermediary step before the actual NGS. The procedure works fine, yet when we wanted to use the same technique on samples of human origin (cell line, tissue or blood), the digestion seems to fail. We are using the same enzyme and buffer batch as for the mouse samples. Yet we do not find back digested fragments for the human samples when doing a size-selection gel, contrary to the digested fragments of the mouse samples that are present. Running the mouse and human samples in parallel, checking the possible interference of reagents used during DNA extraction did not change anything. The conditions of the digestion are the same for both samples, in silico digestion of both genomes seemed fine, and according to literature these enzymes should work for both genomes. Does anyone have any experience/encountered similar problems or has an idea what could be the problem when we shift from mouse to human samples? Thanks for your help!

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Old 07-22-2015, 05:04 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

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That does not make sense. If you can post an image or electropherogram of sample before or better a mock digest (no enzyme) and after digest someone might be able to help.
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