Tru Seq Small RNA preparation kit

[DSP]

Hi!
I am trying to isolate sRNAs from a Total RNA extraction, but I want ALL the RNAs under 200nts (so miRNAs, siRNAs, piRNAs, but also snRNAs and snoRNAs) for sequencing. The Tru Seq kit for sRNA have this way of targeting only RNAs with a certain structure with their specific 3' and 5' adaptors, but how do we know exactly which type of non-coding RNA they include and how could I include all of these above?

Thank you so much!!

David

[kcchan]

The TruSeq Small RNA kit is designed for any RNA that has a 5' monophosphate and 3' hydroxyl. For non miRNA small RNAs, you can treat your sample with Antarctic Phosphatase to remove any phosphate ends followed by Polynucleotide Kinase to add back a monophosphate. You'll also have to adjust your gel step at the very end to account for the difference in size of your sample.

[Genohub]

Be aware that the ligases used in this kit are less efficient as your insert length increases. I wouldn't use a RNA ligase based preparation method for any insert size greater than 50-75bp.

[DSP]

Thanks for your replies! I found a kit called ScriptMiner (Epicentre and Illumina) that says to be able to take even more categories of sRNAs like small 5' capped and 5' triphosphorylated RNAs. Have you ever heard or worked with that kit? I can't find any study which used it in their protocol.

[epibio]

The ScriptMiner Kit includes tobacco Acid Pyrophosphatase (TAP). TAP will convert 5'-capped and 5'-tripphosphate ends to 5'-monophosphate for subsequent 5'-adaptor ligation. the TAP step is optional. By omitting the TAP treatment you construct libraies only from 5-monoP RNA (miRNA, etc). By including the TAP step, you construct libraries from small RNAs with 5'cap, 5'-triP and 5'-monoP ends.
A couple of citations:

Regulski, M et al, 2013 Genome Research http://genome.cshlp.org/lookup/doi/1.../gr.153510.112

Lawlwss, N. et al 2013 PLOS/one
doi:10.1371/journal.pone.0057543

Disclaimer: I work for Epicentre

[DSP]

Hi,
Sorry for the late answer, I was out of town.
Thanks a lot for the information. I had time to read a bit about the Scriptminer protocol and I want to be sure I understand it: If you don't use the TAP step, in the end, it comes back to the same thing as the Illumina's Small RNA Sample Prep kit? Exactly the same?
Thanks again!

[DSP]

The ScriptMiner Kit includes tobacco Acid Pyrophosphatase (TAP). TAP will convert 5'-capped and 5'-tripphosphate ends to 5'-monophosphate for subsequent 5'-adaptor ligation. the TAP step is optional. By omitting the TAP treatment you construct libraies only from 5-monoP RNA (miRNA, etc). By including the TAP step, you construct libraries from small RNAs with 5'cap, 5'-triP and 5'-monoP ends.

Epibio, I guess my question was maybe a bit unclear: if you remove the TAP step, does it only omits 5'-capped and 5'triphosphate sRNAs and goes back to the standard sRNA Illumina preparation kit? Thanks.

[Genohub]

RNA ligase bias

Just want to give you a heads up about the biases associated with RNA ligases.

This article nicely describes the bias issues with Small RNA Seq:

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

- Genohub

[DSP]

Thanks Genohub for the info! But that only gives me another question: how can you get these pooled adapters? Is there kits that already exist or is it too new?

[Genohub]

randomized adapters

I recommend contacting the senior author. He should have recommendations about how to get the randomized adapters. I've heard of some companies working on this, but nothing commercially available just yet.

- Genohub

[Genohub]

Another article on Small RNA-Seq Biases (two kits from the same manufacturer). Looks like it's related to adapter composition:

Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing: http://biorxiv.org/content/biorxiv/e...01479.full.pdf

- Genohub

[NextGenSeq]

We switched to sequencing on the Ion Torrent Proton due to the ligation bias issues. Their small RNA kit from Ambion uses the modified adapters described in the Sorefan paper.

http://www.ncbi.nlm.nih.gov/pubmed/22647250

[CuriousA]

What did you do in the end?

Hi,
I'm planning the same thing: Getting all possible small RNA types that could be found (5'capped, 5'triphosphate, 5'diphosphate, 5'monophosphate).
I will be using the Truseq smallRNA sample preparation kit from Illumina.

I was thinking of using a combination of TAP (tobacco acid pyrophosphatase as it is used in the ScriptMiner protocol) in order to include 5'capped RNAs and some heatlabile phosphatase, for converting 5'tri-di or monophosphates into unphopshorylated 5' end and in the end add a monophosphate with a DNA/RNA kinase.

DSP, what did you do in the end?
Does my plan make sense?

I'm thankful for every suggestion!

[cosmarium]

Hi, I have a question about the product length. I am using True Seq Small RNA preparation kit from Illumina. According to the user guide I should expect a PCR band of about 149 bp (page 23), to be cut off the gel, which corresponds to 125bp adapters + index and my 24-mer sample.
According to the letter to the customer, the lengths are as follows:

5' adapter (part#15013205): 26 nt
3' adapter (15013207): 21 nt

RNA RT primer: 22 nt
RNA 5' PCR Primer: 50 nt
RNA 3' PCR Primer: 63 nt

I am only getting a strong band a little below the 100bp. Is this primer dimer?

My RNA (random 24-mers) plus the two adapters should be 71 nt long.

I will strongly appreciate any insights.

C

[NextGenSeq]

It's hard to tell without you posting a pic of your gel or Bioanalyzer trace. However, if you don't see a band at 140 bp you probably got only adapter dimer.

Not sure why people use the Illumina True Seq Small RNA kit. You will get ligation biased results which are total garbage with that kit.