Small RNA sample prep....

[Sinha_Ashis]

Hello every one....
My name is Ashis Sinha. I am from New Delhi (India). My work revolves around miRNAs and requires sequencing on the Illumina hiseq 2000 platform.

Its nice to meet everyone....

if anyone is familiar with the truseq cluster generation protocol, i would like some information on how much (in pico moles) should i load to have efficient cluster generation, illumina has a feature to discard if there is an overgeneration of clusters...

the protocol says upto 20 picomoles....but i think its too much.

Anything will be of help

[pmiguel]

No, if you overload your lane, then your data quality will suffer and/or the software will be unable to demultiplex you samples.

The problem with answering this question is that "pM" means very little without your specifying what assay method you used to obtain your concentration. The standard answer would be "try 12 pM". But if your stock concentration is inaccurate, then that will not be much help.

--
Phillip

[msheldon]

Hi Ashis,
We do a lot of small RNA Linraries here too, so i know where you are coming from with regard to trying to get optimal clusters. Phillip is right about not overloading. I would recommend using qubit or qpcr to determine concentration. Qbit is pretty good for small rna libraries.