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Old 11-08-2011, 08:08 AM   #1
Location: Europe

Join Date: Jun 2010
Posts: 13
Default library concentrations at various levels (RNA seq)


I am interested in some library preparation details of RNA seq.
Lets assume you deal with GA or HISeq.

I am just interested in the following numbers:

(1) Input Material:
Usually people take 0.1 - 4 ug of total RNA (or, respectively, 100 ng of mRNA). So of what concentration in which volume are we talking about?

(2) Library concentration after amplification (so after purification, fragmentation, first/second strand synthesis, ligation of adapters):
10nM in 10uL .
Would this be a good average estimate?

(3) Library concentration after denaturation and dilution - so when loading it on the flowcell:
7pM in 120uL / lane.
Is this correct?

I would be especially interested in some numbers regarding the TrueSeqProtocol from Illumina.

Thanks a lot in advance,
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