RNA-seq library DSN normalization

megalodon · 03-30-2011, 04:23 PM

Hi,

I was wondering if there are any experienced DSN users out there who would be willing to share their experience with the Illumina DSN normalization protocol.

I have made many Illumina DNA libraries but am in the process of producing my first RNA-seq library from a bacterial metagenomic sample. Reading through Illumina''s DSN Normalization protocol, the procedure seems to be pretty straight forward, however, it looks like there are users who have great success with that method and others cannot get it to work.

I was wondering if anybody who has experience with this procedure would be willing to share their tips and tricks! I am most interested in, e.g., your experience with altering the hybridization temperature and or time as well as DSN concentration used, library concentration used and altering incubation temperature and time for the DSN treatment step. Also, do you have to do the size selection step during the RNA-seq library prep or can that step be skipped since a size selection is performed with the AMPure beads after DSN treatment anyway? Has anybody tried DSN treatment with the new Illumina TruSeq mRNA library prep kit? I have heard that DSN normalization does not work with the new TruSeq kit. If this is true, does anyone know why?

Thank you in advance for your help!

upendra_35 · 04-07-2011, 05:00 PM

Refer this link to answer some of your questions.
http://motif.bmi.ohio-state.edu/ccsb...s%20Report.pdf

ag4lm4 · 09-01-2011, 01:44 AM

Hi,

I am going to try DSN protocol on the preped libraries(Illumina mRNA kit). Post PCr libraries have an additional 60-70bp peak that are primer dimers. I wonder if this peak should be removed by SPRI prior DSN normalization?

Many thanks!

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03-30-2011, 04:23 PM	#1
megalodon Member Location: Pennsylvania Join Date: Sep 2010 Posts: 20	RNA-seq library DSN normalization Hi, I was wondering if there are any experienced DSN users out there who would be willing to share their experience with the Illumina DSN normalization protocol. I have made many Illumina DNA libraries but am in the process of producing my first RNA-seq library from a bacterial metagenomic sample. Reading through Illumina''s DSN Normalization protocol, the procedure seems to be pretty straight forward, however, it looks like there are users who have great success with that method and others cannot get it to work. I was wondering if anybody who has experience with this procedure would be willing to share their tips and tricks! I am most interested in, e.g., your experience with altering the hybridization temperature and or time as well as DSN concentration used, library concentration used and altering incubation temperature and time for the DSN treatment step. Also, do you have to do the size selection step during the RNA-seq library prep or can that step be skipped since a size selection is performed with the AMPure beads after DSN treatment anyway? Has anybody tried DSN treatment with the new Illumina TruSeq mRNA library prep kit? I have heard that DSN normalization does not work with the new TruSeq kit. If this is true, does anyone know why? Thank you in advance for your help!

04-07-2011, 05:00 PM	#2
upendra_35 Senior Member Location: USA Join Date: Apr 2010 Posts: 102	Refer this link to answer some of your questions. http://motif.bmi.ohio-state.edu/ccsb...s%20Report.pdf

09-01-2011, 01:44 AM	#3
ag4lm4 Junior Member Location: UK Join Date: Feb 2010 Posts: 3	Hi, I am going to try DSN protocol on the preped libraries(Illumina mRNA kit). Post PCr libraries have an additional 60-70bp peak that are primer dimers. I wonder if this peak should be removed by SPRI prior DSN normalization? Many thanks!