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Old 07-16-2019, 01:23 PM   #1
Atsushi0803
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Location: Chicago

Join Date: Jul 2019
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Talking Dam-ID prep

Hi all,

I recently sequenced my Dam-ID library. Unfortunately for some reason, I found a lot of read-missing regions even around known binding sites of my target protein. For example, one GATC sequence (this is Dam's target site) right on the binding site of my protein has 10,000 counts, but the GATC 300bp away from the GATC on that biding site has just 200 counts, and vast majority regions have 0 counts. There are some (100~4000) GATC counts in random regions, most of them are not the binding site of my protein and those counts are seen in my soluble Dam contro tool. I also found rDNA got 80% of mapped reads, which is weird because my organism (yeast) has only 10% of rDNA region through out the genome. My protein does not bind rDNA region for sure and the soluble Dam also has 80% of methylation on rDNA region. Do you guys have any thoughts about why I got these biased reads?

Most of my reads started from GATC as I expected and the quality of the reads were fine. I added 25% PhiX to increase the complexity.
I preped Dam-ID library as many people have done: 1. DpnI cut 37oC overnight 2.85oC 20 min 3. Ligation of L-adapter 16oC overnight 4. 65oC 20 min 4. DpnII cut 37oC 4h 5. PCR 6. column purification 7. DpnII cut 8. Column purification 9. Library prep by NEBnext.

I've checked the PCR at step5 was specific to the methylation, as non-DpnI or non-Ligase control was not amplified at all.
Thanks in advance!
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