Hi all,
we're planning to do some metagenomics and metatranscriptomics runs of 5 sediment samples along a depth gradient in a lake, to observe shifts in functioning/expressing and composition with changes in a.o. light and oxygen conditions.
We'd run our libraries on an Illumina HiSeq 2500.
As we don't really have any experiences with this kind of sample preparations (RNA), I've composed a protocol.
The main goal is to target mRNA, but perhaps small RNAs might be interesting too. We want to target the complete community, i.e. eukaryotes, prokaryotes and if possible viruses.
The lake is oligotrophic and a relatively low diversity is present (low complexity of food web, mostly unicellular and few small multicellular eukaryotes)
Could you please comment on the following protocol? Are there better options? Suggestions?
Samples are already taken. LifGuard solution was added for transport (2 months!) and samples were stored at -80°C (also during transport).
Lyophilization of total RNA for TruSeq is not really possible and additionally we don't want to take the risk of possible degradation.
- Extraction:
*MoBio Powersoil RNA extraction kit
(compatible with the Lifeguard solution; removes humic acids which might be present)
*MoBio RNA PowerSoil® DNA Elution Accessory Kit
(we do have separate neighboring cores for metagenomics, but this might be a more sound approach)
- Removal of possible cary-over DNA
Turbo DNA-free
(DNAse is removed with the kit, but is an additional phenol/chloroform step wanted?)
- rRNA depletion
RiboZero Gold epidemiology kit
- cleaning
RNeasy MinElute Cleanup Kit
- cDNA synthesis
Universal RiboClone cDNA Synthesis System
- library prep
Nextera
Are there better alternatives? I'm not very sure abou the cDNA synthesis.
Am I oblivious to some issues?
General hints?
While diversity is relatively low, some depths are dominated by mosses. Does anybody know if it is possible to separate the moss fraction from the rest, as we fear that moss RNA/DNA might engulf the rest?
I'm thinking of first vortexing the samples, pour them over a filter/microsieve, and consequently flush to recover as many epibionts as possible. Or would I jeopardise the RNA integrity?
Btw, we have 5 subsamples/sample of which we would sequence 3 unpooled. One additional subsample would not have the rRNA depleted and also sequenced (but not as deep), so we would have an idea of who is active, without the (additional) bias of the Ribozero kit (as not all rRNA is removed).
Would this be useful?
Thanks in advance!
Kind regards.
we're planning to do some metagenomics and metatranscriptomics runs of 5 sediment samples along a depth gradient in a lake, to observe shifts in functioning/expressing and composition with changes in a.o. light and oxygen conditions.
We'd run our libraries on an Illumina HiSeq 2500.
As we don't really have any experiences with this kind of sample preparations (RNA), I've composed a protocol.
The main goal is to target mRNA, but perhaps small RNAs might be interesting too. We want to target the complete community, i.e. eukaryotes, prokaryotes and if possible viruses.
The lake is oligotrophic and a relatively low diversity is present (low complexity of food web, mostly unicellular and few small multicellular eukaryotes)
Could you please comment on the following protocol? Are there better options? Suggestions?
Samples are already taken. LifGuard solution was added for transport (2 months!) and samples were stored at -80°C (also during transport).
Lyophilization of total RNA for TruSeq is not really possible and additionally we don't want to take the risk of possible degradation.
- Extraction:
*MoBio Powersoil RNA extraction kit
(compatible with the Lifeguard solution; removes humic acids which might be present)
*MoBio RNA PowerSoil® DNA Elution Accessory Kit
(we do have separate neighboring cores for metagenomics, but this might be a more sound approach)
- Removal of possible cary-over DNA
Turbo DNA-free
(DNAse is removed with the kit, but is an additional phenol/chloroform step wanted?)
- rRNA depletion
RiboZero Gold epidemiology kit
- cleaning
RNeasy MinElute Cleanup Kit
- cDNA synthesis
Universal RiboClone cDNA Synthesis System
- library prep
Nextera
Are there better alternatives? I'm not very sure abou the cDNA synthesis.
Am I oblivious to some issues?
General hints?
While diversity is relatively low, some depths are dominated by mosses. Does anybody know if it is possible to separate the moss fraction from the rest, as we fear that moss RNA/DNA might engulf the rest?
I'm thinking of first vortexing the samples, pour them over a filter/microsieve, and consequently flush to recover as many epibionts as possible. Or would I jeopardise the RNA integrity?
Btw, we have 5 subsamples/sample of which we would sequence 3 unpooled. One additional subsample would not have the rRNA depleted and also sequenced (but not as deep), so we would have an idea of who is active, without the (additional) bias of the Ribozero kit (as not all rRNA is removed).
Would this be useful?
Thanks in advance!
Kind regards.
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