Probably this has been covered before, but with the information scattered in numerous threads I'll ask again... What final loading concentrations are people using with a TruSeq stranded mRNA (total fragment size ~ 260 bp) library? We will be using a v3 600 cycles kit. We are aware this is a bit of waste of cycles, as it will read through the adapters. Next time we will probably try to get larger fragments.
We have only done one previous run with a v3 kit (~520 bp fragment). We loaded 9.5 pm and got a cluster density on the low side (784K clusters/mm2). Now the fragment is about half that size; should we load less because of the shorter fragment? This post suggests that shorter fragments will cluster much more efficiently. On the other hand, the previous run was far below the optimum density, so I am inclined to increase the concentration.
So, what final concentrations do you guys load for TruSeq stranded mRNA libraries (with standard fragmentation times)?
Jon
We have only done one previous run with a v3 kit (~520 bp fragment). We loaded 9.5 pm and got a cluster density on the low side (784K clusters/mm2). Now the fragment is about half that size; should we load less because of the shorter fragment? This post suggests that shorter fragments will cluster much more efficiently. On the other hand, the previous run was far below the optimum density, so I am inclined to increase the concentration.
So, what final concentrations do you guys load for TruSeq stranded mRNA libraries (with standard fragmentation times)?
Jon
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