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Old 11-04-2016, 04:20 AM   #1
dr_grm
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Red face Scriptseq complete gold epidem

I have total RNA extracted from environmental samples that I intend to sequence. The main aim is to detect viral and bacterial transcripts.

My lab has purchased the superscript complete gold epidemiology kit for this work. I realized that the protocol epibio/Illumina provide online ends at library QC. I'm confused what steps to follow after that. My questions are..

1. Should I do library normalization or not, and if so, how do I approach this? (can the Nextera bead normalization approach be employed here too?)
2. During library denaturation, should I use NaOH to mix with my libraries while combining heating at 96 for 2 min?

Regards/
dr_grm
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Old 11-04-2016, 01:41 PM   #2
cmbetts
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1) I'm not totally clear by what you mean by library normalization here, but since you reference the Nextera XT beads I'm assuming you mean standardizing the concentrations of multiple libraries so that you get even coverage from multiplexed samples. If so, the answer is yes, but it wouldn't require Nextera beads. They should be treated like a standard library, in which case you can use your preferred method of library quantification (Qubit, bioanalyzer, qPCR, etc.) to calculate the nM concentration of your libraries and then dilute them in 10mM Tris, 0.5% Tween-20 so that you can pool them in equal concentrations for sequencing.
2) The library denaturation protocol is going to be determined by your sequencer. Just about every Illumina sequencer, and even kits for the same sequencer, has a slightly different library prep protocol that should be consulted for the run (Usually NaOH for 5min at RT, sometimes with a Tris quench before further dilution).
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Old 11-14-2016, 03:24 AM   #3
dr_grm
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Hi cmbetts, thank you for your assistance. I have a follow up question. Assuming I have 8 samples I want to multiplex in one run, and I have normalized each sample to 4nM. Should I:
1. Add 5ul of 0.2N NaOH to each sample and incubate for 5 min, then pool 5ul of each denatured sample into 1 tube. or
2. Take 5ul of each sample and pool into 1 tube, then add 5ul of 0.2N NaOH to denature the pool?

Thanks
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Old 11-14-2016, 09:53 AM   #4
cmbetts
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The important thing is to not have any more library or NaOH than used when running a single library. The way that I typically do it is to mix an equal volume of the 4nM libraries together, just enough to accurately pipette, then take 5ul from that pool and treat it like it were a single library (add 5ul NaOH for 5min, then add the HT1 and do any remaining dilutions and/or PhiX additions). I've also seen people denature each library and add HT1 individually followed by mixing in equal volumes prior to loading, but I prefer the first way because it has fewer tubes and steps.
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Old 11-17-2016, 10:03 PM   #5
dr_grm
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Once again, thanks alot cmbetts.
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library normalization, miseq, rna library, rnaseq, scriptseq

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