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Old 08-27-2018, 05:27 AM   #4
UCan'tBcereus
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Location: New England

Join Date: Aug 2018
Posts: 12
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Hi Pete,

Never used the Truseq Kit before, but our lab uses Scriptseq Complete Gold Epidemiology Kit + RiboZero when we make RNASeq Libraries. We use epidemiology kit since we are often working on metagenomic samples; the epidemiology kit removes rRNA for both the bacteria and the host.

Getting high quality Total RNA is difficult with metagenomic samples, but we have successfully made libraries/sequenced of lower quality RNA. Basically, if I see a 16S and 23S peak when QC'ing the total RNA, I can make a successful RNASeq Library. So if RNA Quality is a concern, I wouldn't worry about when using the Scriptseq Complete Gold Epidemiology Kit + RiboZero. High quality RNA works fine too. Our libraries cluster just fine on either the NExtSeq or the Hiseq 2500/4000 when we sequence them.

Quote:
Originally Posted by JakobHedegaard View Post
...
A trick to save even more: The Ribo-Zero kits comes in high or low format for the same price, but the high kit contain app 2x reagent. So we purchases the complete kits in the high version and additional ScriptSeq kits to supply - and follow the low input protocol. Using 500 ng total RNA as input we can hence get two Ribo-Zero reactions out out each High Ribo-Zero reaction.
If you are going to use all 48 ScriptSeq indexes, be aware that index 11 and 31 only have two mismatches to index 41. So use demultiplexing with no-mismatch option.
Best, /Jakob
I completely agree with the above. Good points to keep in mind. Both the "Low Input" and "Regular Input" protocols have worked equally as well for us. To alleviate that indexing issue, you could just make 4 smaller 12 sample pools, instead of making a big 48 sample pool and sequencing across multiple lanes. We usually sequence a 12 sample pool on one Hiseq Lane.


Let me know if you have any other questions.
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