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Old 11-15-2010, 07:19 AM   #1
daughart
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Default Use of DSN normalization in SOLiD RNA-seq?

Ribominus, as many of you know, is pretty labor intensive and inefficient. We are interested in using DSN normalization in construction of SOLiD WT libraries (total RNA, not just poly(A)). DSN seems easier and more efficient in removing all highly-abundant transcripts (more than just rRNAs). However, there's not that much information about how to build DSN WT libraries with Illumina, let alone SOLiD.

My question is, at what step should we insert the DSN protocol? Since at the end of DSN there is an amplification step, taking the purified cDNA into DSN, then going back to size selection and amplification seems like the best solution.

My concerns are that we won't have the correct amount of material for DSN (80-100 ng of DNA), and that there are some other complications we haven't foreseen. If anyone else has thought about this, or has expertise in SOLiD WT or Illumina DSN, we would greatly appreciate any advice.

EDIT: After consultation, it seems like the proper order is to do the entire SOLiD WT library construction protocol, then do DSN, then follow with another amplification round. It would still be great to hear everyone's thoughts.

Last edited by daughart; 11-15-2010 at 07:49 AM. Reason: Update.
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Old 11-15-2010, 08:33 AM   #2
kmcarr
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Quote:
Originally Posted by daughart View Post
However, there's not that much information about how to build DSN WT libraries with Illumina, let alone SOLiD.
Illumina has published a protocol for DSN normalization (see attached application note). The protocol picks up after amplification of the adapter ligated library. They omit the Poly-A selection step of the normal mRNA-Seq library prep protocol (see attached flow chart).
Attached Files
File Type: pdf DSN_Normalization_SamplePrep_Application_Note_15014673_C.pdf (250.9 KB, 63 views)
File Type: pdf RNA-Seq_Flowchart.pdf (32.6 KB, 77 views)

Last edited by kmcarr; 11-15-2010 at 08:38 AM.
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Old 11-15-2010, 08:42 AM   #3
daughart
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Thanks. That order would correspond with DSN after size selection and amplification of the DNA library.

Any thoughts on unexpected complications due to differences between SOLiD and Illumina RNA-seq libraries?
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Old 11-15-2010, 08:45 AM   #4
kmcarr
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Originally Posted by daughart View Post
Thanks. That order would correspond with DSN after size selection and amplification of the DNA library.

Any thoughts on unexpected complications due to differences between SOLiD and Illumina RNA-seq libraries?
Sorry, don't work with SOLiD at all so I'm not familiar with the particulars of their library preps.
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Old 03-09-2012, 12:40 PM   #5
sunkorner
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Hi all, I am planing to use DSN normalization for retrieving good quality gram-negative bacteria mRNA from a sample also containing host plant tissue. We assume that the bacterial quantity is very low. So planning to use of DSN normalization inorder to deplete the rRNA as well as the RNA of the plant from the sample, followed by SOLiD sequencing. Removing the plant RNA as well as rRNA from the bacteria together is the challenge we are facing. Any suggestions will be very useful. Thank you.
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