Weird library with ScriptSeq

cascoamarillo · 01-09-2012, 02:53 PM

Hi,

I'm wondering if anyone else has obtained a result like this in a library construction. Also, a target of this post is the Epicentre tech assist.

I've been contructing a library with ScripSeq Library Preparation kit (Illumina-compatible) from Epicentre. The starting material was rRNA depleted (from Trizol total RNA extraction). The initial RNA and rRNA depleted sample looks good in the bioanalyzer. But after following the protocol and purify the library with XP beads (I also tried MinElute columns, with same result but a lot of primer-dimers), the library pattern does not look good. Attached is the bioanalizer trace from one of the samples (with 15 cycles in the PCR amplification step, having already used between 10-15).

Thanks

Olaf Blue · 01-24-2012, 06:58 AM

Hello-

Can you tell me which product was used to perform rRNA removal, and which ScriptSeq Kit was used to generate the library? Was the original total RNA treated with DNAse I after the trizol extraction? Just some basic questions to gather information to help with finding any issues.

cascoamarillo · 01-24-2012, 07:46 AM

Sure!

I use The RiboMinus™ Eukaryote Kit for RNA-Seq (Invitrogen) to perform the rRNA removal. I work with a fungi, and I've also try The Ribo-Zero™ rRNA Removal Kits (Human/Mouse/Rat) non-magnetic; but with poor rRNA remove.

The kit is The ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-compatible), the version 1.

And no, the original RNA was not treated with DNaseI (it's considered for the future).

Thanks

Olaf Blue · 01-24-2012, 07:58 AM

Hi, thanks for the reply! I'd definitely recommend a DNAse I treatment of your RNA - we've recommended that in the past and it has definitely helped improve the library as it is possible that the cDNA synthesis primer can bind to residual DNA and get extended and thus create a segment of your library.

Rather than using Ribo-Minus or Ribo-Zero, have you thought about using poly-A capture to remove non-mRNA species from the sample? Given the difficulty with the Ribo-Zero and Ribo-Minus Kits with the samples, the mRNA capture might be a better solution.

Just some thoughts.

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01-24-2012, 06:58 AM	#2
Olaf Blue Member Location: Madison, WI Join Date: Nov 2010 Posts: 58	Hello- Can you tell me which product was used to perform rRNA removal, and which ScriptSeq Kit was used to generate the library? Was the original total RNA treated with DNAse I after the trizol extraction? Just some basic questions to gather information to help with finding any issues.

01-24-2012, 07:46 AM	#3
cascoamarillo Senior Member Location: MA Join Date: Oct 2010 Posts: 160	Sure! I use The RiboMinus™ Eukaryote Kit for RNA-Seq (Invitrogen) to perform the rRNA removal. I work with a fungi, and I've also try The Ribo-Zero™ rRNA Removal Kits (Human/Mouse/Rat) non-magnetic; but with poor rRNA remove. The kit is The ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina®-compatible), the version 1. And no, the original RNA was not treated with DNaseI (it's considered for the future). Thanks

01-24-2012, 07:58 AM	#4
Olaf Blue Member Location: Madison, WI Join Date: Nov 2010 Posts: 58	Hi, thanks for the reply! I'd definitely recommend a DNAse I treatment of your RNA - we've recommended that in the past and it has definitely helped improve the library as it is possible that the cDNA synthesis primer can bind to residual DNA and get extended and thus create a segment of your library. Rather than using Ribo-Minus or Ribo-Zero, have you thought about using poly-A capture to remove non-mRNA species from the sample? Given the difficulty with the Ribo-Zero and Ribo-Minus Kits with the samples, the mRNA capture might be a better solution. Just some thoughts.