DNA/RNA hybrid ChIP-seq library preparation

jazz · 11-01-2011, 10:20 AM

Hello everyone,

I am planning to prepare an Illumina library for the DNA/RNA hybridome of the cell. I already have the immuno-precipitated material, and I had a few questions before I begin library prep. I want to proceed in 2 ways-
a) digest the DNA in the sample (leaving just RNA) and perform a standard RNA-seq.
b) digest the RNA using RNase H and prepare the library using the ssDNA as starting material.

My question is regarding the second approach (b). Does anyone have any experience with making libraries using ssDNA as the starting sample?
Any other comments/suggestions are most welcome.

Thanks in advance.

pmiguel · 11-02-2011, 04:54 AM

Not yet, but probably soon. We have a group that is doing a type of IP that yields ssDNA. We have not had our Illumina a year yet, so we are pretty much sticking with the TruSeq kits from Illumina where possible. But we did have success modifying the TruSeq RNA prep protocol to use RiboZero ribo-depleted RNA. We "cut" this RNA into the step immediately following the normal polyA binding step.

So, I was wanting to try a similar trick with this ssDNA. That is, cut it in after the first strand of cDNA synthesis. This may prove to be more difficult. Many of the steps in TruSeq are tightly coupled to increase the speed of the protocol.

Next along those lines would be to replace the nucleotide mix used in second strand synthesis with a dUTP-instead-of-dTTP dNTP mix. That way, after adapter ligation, a uracil-N-glycosylase step could be added to produce strand-specific libraries.

--
Phillip

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11-01-2011, 10:20 AM	#1
jazz Member Location: us Join Date: Nov 2008 Posts: 28	DNA/RNA hybrid ChIP-seq library preparation Hello everyone, I am planning to prepare an Illumina library for the DNA/RNA hybridome of the cell. I already have the immuno-precipitated material, and I had a few questions before I begin library prep. I want to proceed in 2 ways- a) digest the DNA in the sample (leaving just RNA) and perform a standard RNA-seq. b) digest the RNA using RNase H and prepare the library using the ssDNA as starting material. My question is regarding the second approach (b). Does anyone have any experience with making libraries using ssDNA as the starting sample? Any other comments/suggestions are most welcome. Thanks in advance.

11-02-2011, 04:54 AM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Not yet, but probably soon. We have a group that is doing a type of IP that yields ssDNA. We have not had our Illumina a year yet, so we are pretty much sticking with the TruSeq kits from Illumina where possible. But we did have success modifying the TruSeq RNA prep protocol to use RiboZero ribo-depleted RNA. We "cut" this RNA into the step immediately following the normal polyA binding step. So, I was wanting to try a similar trick with this ssDNA. That is, cut it in after the first strand of cDNA synthesis. This may prove to be more difficult. Many of the steps in TruSeq are tightly coupled to increase the speed of the protocol. Next along those lines would be to replace the nucleotide mix used in second strand synthesis with a dUTP-instead-of-dTTP dNTP mix. That way, after adapter ligation, a uracil-N-glycosylase step could be added to produce strand-specific libraries. -- Phillip