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Old 07-15-2013, 12:31 AM   #1
lihzhou
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Location: China shanghai

Join Date: Apr 2013
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Default Illumina library construction from ssDNA (MeDIP)??

Hello everyone,

As the original protocols for this DNA methylation sequencing library construction (MeDIP-seq) basing on following procedures: 1) shearing of gDNA, 2) End repair, 3) A-tailing, 4)adapter ligation, 5) denaturing of DNA, 6) MeDIP, 7) PCR enrichment

From now on, we have tried so many MeDIP-seq libraries (ideal concentration and distribution in Agilent 2100) but failed in qPCR detection of the MeDIP enrichment of the library( Δ Ct of the negative and positive almost below 1.5, even worse for Rat gDNA samples). When we directly progress MeDIP with the gDNA, this problem dissapeared ( Δ Ct over than 5). It seems that adapter ligation dramatically influences MeDIP enrichment.

So does anyone know what's the reason of my problem??? As the adapter we used is not methylated.

Other question is that it maybe a resolvent to do MeDIP first and then construct libraries. Anyone knows the protocol for library construction from ssDNA??


Best regards
-LHZ
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Old 07-25-2013, 06:12 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Quote:
Originally Posted by lihzhou View Post
Hello everyone,

As the original protocols for this DNA methylation sequencing library construction (MeDIP-seq) basing on following procedures: 1) shearing of gDNA, 2) End repair, 3) A-tailing, 4)adapter ligation, 5) denaturing of DNA, 6) MeDIP, 7) PCR enrichment

From now on, we have tried so many MeDIP-seq libraries (ideal concentration and distribution in Agilent 2100) but failed in qPCR detection of the MeDIP enrichment of the library( Δ Ct of the negative and positive almost below 1.5, even worse for Rat gDNA samples). When we directly progress MeDIP with the gDNA, this problem dissapeared ( Δ Ct over than 5). It seems that adapter ligation dramatically influences MeDIP enrichment.

So does anyone know what's the reason of my problem??? As the adapter we used is not methylated.

Other question is that it maybe a resolvent to do MeDIP first and then construct libraries. Anyone knows the protocol for library construction from ssDNA??


Best regards
-LHZ
We had good results for a customer making normal TruSeq DNA libraries -- but with non-methylated adapters. The customer did the MeDIP then we amplified the precipitated DNA.

For a simple way to make a library from ssDNA, just use an RNA seq library method, like the original TruSeq RNA prep library, starting at the step after 1st strand cDNA synthesis.

--
Phillip
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Old 07-27-2013, 04:42 AM   #3
Genohub
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A few PCR cycles after adapter ligation will ensure double stranded DNA. Then skip your denaturation of DNA and directly enrich from double stranded product. This tends to work well. Are you doing any size selection after ligation? A significant portion of your material going into enrichment could be adapters or adapter dimers.
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