SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq: High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Newsbot! Literature Watch 1 01-22-2013 12:48 AM
RNA-Seq: A Strand-Specific Library Preparation Protocol for RNA Sequencing. Newsbot! Literature Watch 0 09-29-2011 07:00 AM
SOLiD 4 ChIP-seq library preparation ETHANol SOLiD 1 05-05-2011 07:28 AM
RNA-Seq: Method for improved Illumina sequencing library preparation using NuGEN Ovat Newsbot! Literature Watch 0 04-14-2011 03:50 AM
how much DNA is required for library preparation? chromatin Sample Prep / Library Generation 0 11-14-2010 07:46 PM

Reply
 
Thread Tools
Old 11-01-2011, 10:20 AM   #1
jazz
Member
 
Location: us

Join Date: Nov 2008
Posts: 28
Default 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.
jazz is offline   Reply With Quote
Old 11-02-2011, 04:54 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

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
pmiguel is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 07:57 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO