Dear worm breeders,
This is Jian Chen from Tim Schedl lab at WashU.
Can anyone recommend a kit to remove rRNA from total RNA samples?
Any ideas what to use to prep RNA-seq from SMALL amount of RNA (1-10ng)? I heard nice things about SMART-kit from Takara, anyone tried before? Any better options?
Thanks,
Jian
Hi Jian,
I’ve been pretty successful doing RNA-seq from FACS sorted neurons (<10ng RNA) using NucleoSpin RNA XS in combination with SMART-Seq v4. The NucleoSpin columns work much better than the Qiagen RNA kits.
Thanks a lot, Snug.
I just did one round of sample harvest, and surprisingly I can get 80-300ng of total RNA (determined by Qubit, not Tapestation or Bioanalyzer). It will be great to have rRNA removed, rather than getting the mRNA only. Any recommendation on rRNA removal kits works for you?
Jian
Hi Jian,
That’s a lot of RNA! Definitely run it on a Tapestation or Bioanalyzer next to check your RIN. I’ve actually just gone straight to SMART-Seq prep after as it should select for mRNA. I have heard of a Qiagen kit that’s been optimized for worm rRNA removal from Qiagen. It’s kinda expensive, but you can request a trial kit (link is on the product page) for evaluation.
Hi Snug,
Thanks a lot for your suggestions. Definitely will give it a try.
Jian
Hi Jian, I’ve also been doing a lot of work with FACS sorted neuron samples, with 0.5-10ng RNA input for RNAseq libraries generally. We directly compared the SMARTseq V4 kit (polyA selection) to the SoLo Ovation kit from Tecan Genomics (targeted Ribodepletion) on matched neuronal samples sorted using a rab-3 driven GFP marker.
Tecan doesn’t usually make a C. elegans rRNA probe set so we got them to make a custom set of probes.
Our comparison is published in G3 PMID: 33856427 (I’m a new user so I can’t post a link)
Overall take: Both kits gave a similar “useful reads per million” although smartseq has better rRNA exclusion. Predictably the SoLo kit retains non-polyA noncoding RNAs that are lost in SMARTseq. The SoLo kit gives more uniform gene coverage. The SoLo kit shows lower gene level variance, which is potentially important as all the samples are from the same tissue. And the SoLo kit seems to show better detection of long genes.
I haven’t tried the Qiagen kit.
The SoLo ribodepletion kit also has UMIs, which makes deduplication more accurate, which I don’t see in the Qiagen kit (on first pass at least).
-Alec
Hi Alec,
Thanks for the information. It’s always good to know that you have other options. Sorry that I haven’t check the messages on wormbase for a while. Thanks a lot for generating the side-by-side comparisons for these two kits for me.
I have sent an email to the company, asking for the pricing for the kit and specifics on how to order their customized rRNA probes mentioned in your paper.
To maximize the flexibility of the experiments (so that the kits/protocols can handle variable amounts of input RNA, different types of RNAs sample sources: direct isolated total RNA or other fragmented RNA), I always like to treat the lib prep in two different steps: rRNA depletion and the actually lib prep. And use the best options available to me.
I have one question about the primers used in the first strand synthesis. The SMART-v4 used dT and the Solo uses dT and random primer. My choice of primer is usually random primer over dT for less bias between 5’ and 3’ end coverage, if I have the choice. But in actual fact, I don’t know if the combination of dT and random outperforms random primers only. What do you think?
Thanks,
Jian
Hi Jian,
Yeah this approach is rRNA depletion after fragmentation and initial cDNA library amplification, so not as flexible as the ideal… More traditional approaches like RNase H with c elegans specific probes (such as the ones developed by the Fire lab a few years ago) may also work if you’re consistently getting > 100ng of high quality RNA. But for our single neuron type sorts we have a really wide range of yields, down into the single digit nanograms for a few thousand cells.
I don’t have a sense of whether the dT plus random primers outperforms random primers on their own (I’ve never tried a truly random primer set), but we got quite uniform gene coverage with the SoLo kit. I can come up with justifications for why it might work, but none of them are backed up by actual data.
-Alec Barrett
Hi Alec,
So I just talked to Tecan, and the anydeplete is really impressive. They are sending me some samples over for me to try out. The only issue is that I can’t QC the rRNA depletion before the sequencing.
The kit ask for 10ng library DNA to enter the anydeplete, and the whole process, how much library DNA do you usually have? I see an amplification step is involved, do you know what that might be? Isothermal amplification?
Jian
Hi Jian,
I work with 0.5ng to 5ng of total RNA prior to library prep, and after all steps I usually have ~40-70 ng of cDNA in the sequencing library, which is plenty to sequence at depth. I’d also note that there are 2 steps where you have a cDNA library, first after fragmentation and ligation you amplify and get a cDNA library (let’s call it lib 1) that includes rRNA sequences, then you deplete and amplify again and get the sequencing library (lib 2).
For lib 1 I almost always have more than is needed for amplification according to the kit, so in addition to having plenty of cDNA in lib 2 for sequencing, I have a backup of non-depleted cDNA that I can fall back on if I need more sequencing for whatever reason.
-Alec Barrett