New ScriptSeq v2 RNA-Seq Library Prep Kit

[epibio]

The new ScriptSeq™ v2 RNA-Seq Library Preparation Kit from Epicentre is now available. The kit offers several improvements over the original ScriptSeq Kit, including better transcript coverage, lower RNA input requirement, a streamlined procedure, and lower cost. For more information, please see the product page.

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[Dario1984]

Are there UCSC Browser tracks of mapped reads anywhere of a whole sample dataset ? In other words, not just a screenshot of one gene, which all the kit manufacturers seem to think is convincing. We're interested in a kit with good 5' to 3' evenness for mRNA-seq. The RNA is from cell lines and is not degraded.

[epibio]

Not yet, but we're working on it. At present, the other data we have is a 600-gene coverage plot that I can send you.

[ZWB]

I would also be very interested to see your 600-gene coverage plot. Thanks

[epibio]

This is the 600-gene coverage, single-end sequencing reads from the 5' end so there is an expected drop in coverage at the 3' end. I'll post the UCSC browser plots when they are available.

[censinis]

Based on manufacturer's suggestion, the ScriptSeq v2 RNA-Seq Lib Prep kit is well suited to work with 500 pg to 50 ng of either ribodepleted or poly(A)+ RNA. I am wondering if it is possible to start with totRNA of bacterial origin and, in that case, how much totRNA has to be reverse transcribed.
Best
S

[yoon]

Do you have data of how this kit perform with other mainstream kits? truseq RNA will be nice if you have a comparison

[epibio]

Originally Posted by censinis

Based on manufacturer's suggestion, the ScriptSeq v2 RNA-Seq Lib Prep kit is well suited to work with 500 pg to 50 ng of either ribodepleted or poly(A)+ RNA. I am wondering if it is possible to start with totRNA of bacterial origin and, in that case, how much totRNA has to be reverse transcribed.
Best
S

In theory, you could use the kit with total RNA at the higher end of the input range. However, since 95-99% of your reads will map to ribosomal RNA, it would not be a good idea.

[censinis]

Of course. Nevertheless, I did experience not exciting results in ribodepleting meningococcal rRNA from pretty good totRNA preps (RIN >9), with approx 60% of rRNA related reads still left after Ribo-Zero (Gram-) treatment as judged by evaluating HiSeq 2000 output data. Because the enormous mole of sequences we can raise in a single HiSeq lane (approx 30Gb), we can still expect enough space to approach the expected bacterial transcriptome complexity. Please let me know if you have any any alternative suggestion.
Thanks in advance
S

[Jperrott]

I was wondering how well would the ScriptSeq V2 kit work on roughly 1ng total RNA? I ask this because performing a Ribominus or PolyA selection on such a small amount of total RNA seems risky. I realize that we would most definetly get rRNA reads mapping, but even with a 40% mapping of mRNA fragments, this would be ideal for my particular application.

[epibio]

The ScriptSeq v2 kit will perform well with 1 ng total RNA; as you expect, the majority of your reads would be ribosomal RNA.