Ribo-Zero before or after DNase treatment?

[colin.aibn]

Hi everyone,

I have been looking at a few different protocols to process RNA samples for RNA sequencing and I am a little confused about the order of some of the steps in the process.

I have a gram-negative bacteria that I have extracted RNA from using an RNeasy midi kit. I would now like to digest the DNA and deplete the 23s/16s, however, I'm not confident with which step should be done first as some protocols I have read perform rRNA removal before DNase treatment. This could be problematic because, from what I have read about rRNA removal from even large amounts of RNA initially (~5 ug), the yield can be quite low (100 -> 300 ng). It seems it would be better to first perform the DNase digestion on the RNA and get a decent yield and then rRNA deplete the sample. Otherwise, if you do it in reverse and perform the DNase digestion last you risk losing a larger percentage of your already low yield.

Does anyone have any suggestions as to which step is best to do first?

In which order do people normally do these steps?

[protist]

DNase first and then rRNA deplete. Ensure that your DNase has been effective before progressing on to library prep.

[colin.aibn]

Sounds good, thanks for your help.

[colin.aibn]

One more question, should I precipitate and resuspend the RNA after the DNase digestion but before the rRNA depletion or just rely on the precipitation/resuspension at the end of the rRNA depletion to remove unwanted carry over like EDTA?

[protist]

EDTA should not be a problem as some protocols recommend re-suspension of RNA in TE or water. The main problem would be salts (e.g., Mg2+ or guanidinium salts) or organics (e.g., phenol and ethanol). Dependent on the method you used for DNase you volume may be too large for you depletion method thus a precip or concentration would be required. Post DNase we use Zymo Clean&Concentrate RNA 5ug columns, in fact we use the columns for concentrating the fragmented RNA also - it has made a huge difference in yields especially for our bacterial RNAseq libraries.

[colin.aibn]

Thanks again protist.

I was planning on using invitrogen's amplification grade DNase I for two digestions (unless of course the first digestion is good enough). What I might do, instead of doing a precipitation between digestions, is use the zymo clean & concentrate column to reconcentrate the first digestion and then take the second digestion directly into the ribo-zero degradation and clean it up at the end, as the ribo-zero kit recommends, with the zymo clean and concentrate kit.

Do you see any problems with doing that?

[protist]

Thanks again protist.

I was planning on using invitrogen's amplification grade DNase I for two digestions (unless of course the first digestion is good enough). What I might do, instead of doing a precipitation between digestions, is use the zymo clean & concentrate column to reconcentrate the first digestion and then take the second digestion directly into the ribo-zero degradation and clean it up at the end, as the ribo-zero kit recommends, with the zymo clean and concentrate kit.

Do you see any problems with doing that?

Sounds good to me - although you may not need to clean-up in between your two DNase steps.. There are various flavours of Zymo RNA columns - the 5 ug are good for up to 5 ug RNA and you can elute in as little as 6 uL but I also use the 25 ug RNA column variants for larger scale RNA preps. We use Roche/USB recombinant DNase and either phenol extract or pass through RNA column for clean-up. We check our RNA with a quick PCR to check for DNA contamination before proceeding with library prep - we learned the hard way when a set of our bacterial libraries turned out to be more reflective of genome than transcriptome! We have only converted to RiboZero in the last few months and I have to say it is brilliant. Are you using the metabacteria or the Gram negative kit?

[colin.aibn]

I'm using the gram negative kit. Glad to hear its working well for you.

How many amplicons do you look at in your pcr and what length are they? I was going to run a pcr before DNase treatment and after treatment so I can see how the digestion works using 3 amplicons of about 60 to 70 nt. I'm also making sure that the EDTA conc. from the inactivation step stays between 1.5mM and 2 mM