[pmiguel]

Hi,
Have you seen artifact sequences "CCACGTTCCCGTGG" in TruSeq Small RNA system?
I often found this sequence in TruSeq SmRNA system, not in old Small RNA System (v1 & v1.5).

Did you ever find out what this is? I am seeing it too in my TruSeq smRNA libraries.

I don't know what it does, but:

It matches the "Stop Oligo (STP)" sequence in the Illumina Customer Sequence Letter. "STP" is the three letter code for "Stop Solution" in the TruSeq Small RNA prep kit. Added as the last part of the T4 RNA Ligase2 (deletion mutant) ligation of the 3' adapter to the RNA. The sequence also has the footnote "patent pending" in "the Letter". Not sure what patent that would be.

--
Phillip

[vtosha]

NEB kit use heat inactivation of ligase (see instruction to ligase). As I think Illumina use NEB-reagents for it's sample prep kits, so I try to change Illumina manual and use temperature inactivation instead Stop Solution and I had normal libraries.

[beibeizhou]

truseq small RNA library

how many library yield can you get using truseq small RNA kit ?

[vtosha]

1-20 nM. We use a half dose of reagents.

[katjef]

We have ribosomal RNA-depleted samples that we want to examine both long RNA (mRNA, lncRNA) as well as small RNAs. Do we need to prepare 2 separate libraries for small and long RNA? Or would a Truseq (Illumina) library prep be sufficient to get all RNA types?

[Phage434]

That sequence is from the 3' end of "Blocker 4" in this patent:

http://www.google.com/patents/US8575071

It's used to block ligation of the excess adenylated 3' linker.

[vtosha]

We have ribosomal RNA-depleted samples that we want to examine both long RNA (mRNA, lncRNA) as well as small RNAs. Do we need to prepare 2 separate libraries for small and long RNA? Or would a Truseq (Illumina) library prep be sufficient to get all RNA types?

You can but you should to treat them separately. Use Qiagen kit for small RNA preparation, it help to get both types of RNA.
You can use one kit Truseq Small RNA for both types of libraries but long RNA need to additional treatment with Antarctic phosphatase and kinase. You can find this protocol on Illumina site.

[windhorse8]

What I did was to size select the RNA fraction: 15-20% PAGE in denaturing contitions, cut the gel band (15-25 or 20 -35; it depends what it's for); elute ON in salt solution and precipitate. And the size selected RNA is used for the library construction (3' ad ligation, etc...). Hope it helps.

Do you construct the library (3' adapter ligation and so forth) using small RNA sample prep kit or using your own adapters, primers, and other reagents. I am also making libraries but I have to use adapters and primers that are not from the kit. Does it matter ?