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Old 09-03-2014, 08:52 AM   #1
RNAarecrazy
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Location: Massachusettes

Join Date: Jan 2014
Posts: 4
Default ssDNA library prep for meDIP

Hi everyone, I am working on meDIP-seq using antibody that recognizes methylated cytosines to pull down methylated regions in the genome.

I did it a bit differently from conventional protocols, in that I prepared library from the single stranded DNA pulled down by meDIP. It seemed to have worked. Here is what I did:

1) perform meDIP on denatured, fragmented gDNA.
2) convert the single stranded me-DIP'ed DNA to dsDNA by random primer and DNA polymerase (basically the second strand cDNA synthesis in standard RNA library prep)
3) Carry through with end repair, dA tailing, adaptor ligation, amplification

The end result: beautiful library with differential enrichment in methylated versus non methylated regions.

The problem: no one has done this before. Usually people add the adaptors onto the gDNA BEFORE meDIP when the gDNA is still double stranded. I found that this reduces IP efficiency.

Since my method is not the standard, there must be something wrong! Can some one tell me why it is not the main stream thing to prepare library from ssDNA in meDIP????
thanks a bunch.
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Old 09-08-2014, 07:51 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Quote:
Originally Posted by RNAarecrazy View Post
Hi everyone, I am working on meDIP-seq using antibody that recognizes methylated cytosines to pull down methylated regions in the genome.

I did it a bit differently from conventional protocols, in that I prepared library from the single stranded DNA pulled down by meDIP. It seemed to have worked. Here is what I did:

1) perform meDIP on denatured, fragmented gDNA.
2) convert the single stranded me-DIP'ed DNA to dsDNA by random primer and DNA polymerase (basically the second strand cDNA synthesis in standard RNA library prep)
3) Carry through with end repair, dA tailing, adaptor ligation, amplification

The end result: beautiful library with differential enrichment in methylated versus non methylated regions.

The problem: no one has done this before. Usually people add the adaptors onto the gDNA BEFORE meDIP when the gDNA is still double stranded. I found that this reduces IP efficiency.

Since my method is not the standard, there must be something wrong! Can some one tell me why it is not the main stream thing to prepare library from ssDNA in meDIP????
thanks a bunch.
Well, if there is a "heavily" used protocol it tends to be incumbent upon someone developing a different protocol to show the new protocol works and give some idea as to how results from the new protocol compare to results from the old protocol.

I tend to agree with you that doing IP on the genomic DNA prior to ligating adapters on makes more sense.

--
Phillip
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