[epibio]

Mycoplasma contamination will cause problems with any random-primed library prep method, due to the relative abundance of the mycoplasma RNA.

@gao331: If you are not sure about the ScriptSeq kit, we do have an evaluation program. Please PM me for details.

[JStephans]

Works well, reads are oriented off the negative strand and will appear as blue for positive and yellow for negative reads in GS.

[upendra_35]

[QUOTE=spenserbsmith;31174]I have run the scriptseq with both Poly A+ enrichment and the ribozero kits. So far, neither has worked at the level we hoped for; however, I'm am new at generating libararies for sequencing, so I'm not totally confident in my results. A close colleauge of mine has done a full RZ+SS run and got good libraries, but still with more reads of Ribosomes than epicentre got in their results.

Hi i have recently tried to use Scriptseq for making RNAseq libraries but unfortunately none of my libraries worked with the kit. I am wondering if there are modifications you made for the Scriptseq kit? Any help would be appreciated.

[upendra_35]

Hi i have recently tried to use Scriptseq for making RNAseq libraries but unfortunately none of my libraries worked with the kit. I am wondering if there are modifications you made for the Scriptseq kit? Any help would be appreciated.

[epibio]

@Upendra_35: Please contact our technical services scientists at 1-800-284-8474 so we can discuss why you couldn't get the kit to work. There are many issues that affect the quality of a RNA-Seq library, so it would be easier to determine the problem if we had detailed information.

[RNAseqer]

We do several 100 mRNA libraries ourselves and have been looking into kit alternatives for some time. The scriptminer kit from epicenter has failed in our hands. We couldn't get libraries. Right now we have our own method similar to the illumina methods and that works well for us.

Hi i have recently tried to use Scriptseq for making RNAseq libraries but unfortunately none of my libraries worked with the kit. I am wondering if there are modifications you made for the Scriptseq kit? Any help would be appreciated.

[RNAseqer]

Sorry meant to say that the scriptseq kit for mRNA has failed our rigorous testing.

[upendra_35]

Thanks RNAseqer for the update on Scriptseq kit. Is it possible for you to share your in-house protocol for RNAseq library making? Thanks in advance

[lvandiepen]

I'm new to this discussion forum, but found this thread very interesting.
I am also looking for alternatives to the mRNA library prep kits that are so expensive.
RNAseqer and/or upendra_35, I am also very interested in the in-house protocol for RNAseq library making!
What type of samples are you applying it to? I want to use it on soil RNA extracts.
Thanks so much!
Linda

[upendra_35]

I'm new to this discussion forum, but found this thread very interesting.
I am also looking for alternatives to the mRNA library prep kits that are so expensive.
RNAseqer and/or upendra_35, I am also very interested in the in-house protocol for RNAseq library making!
What type of samples are you applying it to? I want to use it on soil RNA extracts.
Thanks so much!
Linda

We use Plant tissues for making RNA libraries. We are in the process of publishing the in-house protocol for RNAseq libraries very soon. Once its been published or been accepted for publication i can send you the link. Will keep you updated on that. The good thing about this protocol is the extraction of mRNA directly from plant tissues and proceeding to library making. It gave satisfactory results.

[serdarumd]

Hi,
I have recently made a sequencing library using Epicenter's ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina-compatible). Now I am confused about which Illumina kit I am going to use to continue to get sequencing results. The guys at Epicenter could not tell me anything. They told me one primer (RD1 SP) for sequencing is from one Illumina kit and the other primer is from another Illumina kit, and that is it. Can anyone help me with this? Thanks a lot.

[epibio]

Please see "Appendix 2: Sequencing the ScriptSeq (Illumina-compatible) Library" in the protocol on page 14 for full details. Briefly:

The Read 1 Sequencing Primer (Rd 1 SP) is included in the Illumina Standard Cluster Generation Kit v4 (Illumina catalog number GD-103-4001). The Read 2 Sequencing Primer (Rd2 SP) and the Index Read Sequencing Primer (Index SP) are included in the Illumina Multiplexing Sequencing Primers and PhiX Control (Illumina catalog number PE-400-1002).

[serdarumd]

Hi,
Yes I have read the appendix and I know where to find those primers. My question is now that I have my library that I need to sequence which Illumina kit or kits can I use to sequence my library?

[epibio]

Assuming that you have a GAIIx instrument, you would use the standard cluster generation kit v4 and Illumina sequencing kit v4.

[serdarumd]

Thanks for the quick reply. That helped a lot.