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
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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