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I take 1 ug of total RNA and ribo deplete it. The leftover is usually 15 % of the total RNA. I am using reagent quantities as prescribed. May I ask how much Input you use??
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250ng-1ug, RIN>8Comment
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Hi,
I have 151 nM/ ul library concentration. I diluted it to 1nM/ul by adding 1 ul to 150 ul water. Now if I take 10 ul from this (1nM/ul) will it be equal to 10 nM/10 ul.
Or I should dilute 10nM???Comment
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Your desired final concentration of your library will depend on what Illumina sequencer you're using and what the facility calls for. Will you be sequencing this library yourself?
For reference, Illumina recommends storing libraries at either 2nM (HiSeq/NextSeq) or 4nM (MiSeq) and diluting to the appropriate molarity before clustering. I generally store my libraries at >10nM as I see a gradual drop in molarity over time corresponding adsorption to the plastic storage tube -- this can be mitigated by using "LoBind" tubes and adding Tween to your storage buffer.Comment
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Thanks, adam.geber for you reply.
Sequencing is done by Service provider and they asked me to pool the samples to 10nM for HiSeq 3000. I will consider storing the samples as suggested..Comment
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Definitely a good idea to store at a higher concentration than needed for submission. For reference, you'll need to redilute your 151nM library to 10nM with e.g. 5uL of your library in 70.5uL of dilution buffer or whatever volumes work best for you. Illumina recommends Tris-Cl 10*mM, pH*8.5 with 0.1% Tween 20 rather than water.Comment
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Thanks adam.geber. I don't know but I was asked to send samples in MQ water.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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