[smalan]

Hi guys, has anyone used the ScriptMinor kit from this company, for sequencing small amounts of miRNAs? I want to know if it is worth my while to order this.

[mmark27]

BUMP on this. I think we should be using the fr-firststrand according to this explanation?

http://www.epibio.com/faq/ScriptSeq_RNA-Seq_Library_Prep_Kits.asp

Library Type Examples Description
fr-unstranded Standard Illumina Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
fr-firststrand dUTP, NSR, NNSR Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
fr-secondstrand Ligation, Standard SOLiD Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

I know this thread is wicked old, but we're using the Scriptseq V2 kit and we see the same thing on our data when loaded into the Genome Browser. Everything is shifted slightly. Epibio didn't have an explanation of why.

What are people using during TopHat for library type? fr-firststrand, fr-secondstrand????

ALSO, what about the shifting on the display in UCSC as below? We see it too, I've heard no good explanation.

Dear all,

I am analyzing the result of RNAseq on Hela cells. Libraries were created by Epicentre ScriptSeq kit, and they have around 7.000.000 reads each. I mapped the reads with TopHat to both hg18 and hg19. Most of transcripts look OK, but weird thing happens with RNA genes-snRNA, snoRNAs etc. Almost all reads cover reference RNAs starting from nucleotide +5 to +10, not from the annotated 5' end, even if the total coverage is very high. Out of more than 2000 reads mapped to U1 snRNA only 2 or 3 have their 5' end identical to the gene. Have anyone experienced this? Is there some technical issue with the library construction or sequencing, or a mapping artifact?
Best,

Aleks

[[email protected]]

dear colleagues,

We are currently using the ScriptSeq version 2 kit to generate RNA-seq libraries for a study.

For this study we have a total of 127 samples that we wish to individually barcode and divide into 3 pools for sequencing, with each pool containing ~42 individually barcoded samples.

We were therefore wondering if it is possible to use 42 of the 48 barcodes currently available from Epicentre (from primer sets 1-4) to generate these 3 pools or are there certain barcode combinations that cannot be included in the same pool due to nucleotide sequence similarities in the barcodes that render deconvoluting of the pools difficult?

Many thanks

Dave

[jtyler]

I have a related concern as Jagruh last year:
epibio: Is ScriptSeq mRNA-seq library prep kit (for Illumina) strand specific? From the mechanism, it seems strand specific, but in our sequenced result there are still enormous hits to the other strand. We wonder whether these anti-transcripts are biological (in bacteria) or artifacts.
We see a huge amount of antisense reads on yeast total RNA seq. Can you send me links to show me what your kit gives for strand specific RNA reads in your hands.
Do you know why we are having this problem?

[diagen]

ScriptSeq cDNA synthesis implies a random priming (tagged). It can be strand-specific only in case if their reverse transcriptase StarScript is not processing DNA templates. Since there is no solid statement on such unique properties (surely they did such tests) it likely works as DNA polymerase, but probably with less processivity than superscript. Unlikely they added actinomycin or so because it will interfere with the next step utilising Klenow activity. So I would expect some leakage to the opposite strand occur on highly abundant transcripts.

[c.ava]

Dears,
I need to kwow the mechanism wich we obtain the directionality in RNA libraries with ScriptSeq v2. Can you help me? Pleaseeeee

[MfA]

Dear all,

I will start next week with a new RNA project. For that I choose the ScriptSeq RNA Seq Kit v2 for library preparation. I will do poly A purification and starting amount for the library will be ~50ng. Now I am wondering at the number of amplification PCR cycles. 10 Cycles seem for me like over-amplification. Does anyone has experience with similar conditions??? In the past I used a homebrew method (Fragmentation, cDNA, E-Repair, A-Tailing, Adapter ligation, Size selection) where I had mostly only 10-20ng of poly A RNA as starting amount. And for those samples it was more than enough to use 8-10 cycles for amplification. Any suggestions???

[NeleZ]

50 ng/ul is plenty! One ng of library is sufficient for an entire flow cell (~500 million reads on a HiSeq).

I also get very low yields for my library after 15 cycles of PCR (~5-15ng/ul) although I start with ~4ug total RNA i.e. ~100ng polyA. Same for others in our lab.
This seems really low to what other users get and I wonder how that could be?
My specific problem is that I still want to size-select for a relatively narrow range (100bp range) and thus will be left with very little amounts making it hard to get a reliable quantification...

[MfA]

When you are doing the Size Selection??? After amplification? And from 4µg total RNA you got 100ng poly A RNA or 100ng ribo-depleted RNA?
5-15ng/µL will be more than enough for sequencing. You only need a 10nM library and this you should have with 5-15ng/µL.

[NeleZ]

When you are doing the Size Selection??? After amplification? And from 4µg total RNA you got 100ng poly A RNA or 100ng ribo-depleted RNA?
5-15ng/µL will be more than enough for sequencing. You only need a 10nM library and this you should have with 5-15ng/µL.

I do the size selection after the amplification and I use polyA selection

[MfA]

So I used 50ng poly A as starting material and got at the end concentrations 12-25ng/µL, but without size selection. But I know, that with size selection you really loose a lot of material. So I would suggest that 5-15ng/µL final concentrations are good values for libraries with size selection. Don't worry about this. If you are not satisfied with this, you can have a trial for size selection with ampure beads. But this only works, if the size range is not that big.