2. Validate the final density using a refractometer and adjust accordingly if the reading differs from nD-TC = 1.4031 +/- 0.0002.
   3. Balance each tube pair according to the instructions of the ultracentrifuge's manufacturer.
   4. Centrifuge at 177,000 \(\times\) gav (49,500 RPM for the VTi 90 rotor) at 20 °C for >36 h at maximum acceleration and minimum deceleration (no brake). 

3.1.2 Preparation of gradient mixture for two-step DNA-SIP using bis-benzimide

  1. Collect and pool fractions corresponding to densities between 1.725--1.735 mg ml-1 (see section \ref{787481}) and discard the rest.
  2. Recover the DNA from these pooled fractions through precipitation. Resuspend in about 20--30 µl of TE (see section \ref{343649}).
  3. For the secondary centrifugation gradient, prepare a fresh CsCl gradient by following the steps above (see section \ref{434098}), but replace 8 µl of the GB with 8 µl of bis-benzimide (10 mg ml-1).
  4. Load the recovered DNA from the first centrifugation step. 
  5. Proceed with centrifugation, fractionation and DNA recovery as usual for DNA-SIP (see sections \ref{434098} and \ref{343649}).
  6. The fractions corresponding to densities between ca. 1.690 and 1.710 mg ml-1 should now contain the labelled DNA while fractions corresponding to densities between 1.710 and 1.713 mg ml-1 should contain unlabelled high-G+C DNA.

3.1.3 Preparation of gradient mixture for RNA-SIP

  1. For each gradient, mix GB, RNA sample (300--500 ng), and CsTFA stock solution in a separate 50 ml tube according to equation \ref{eqn:mix2} for a final density of 1.825 g ml-1, but using only 97% of the final volume to leave room for the formamide (below). Assuming 4.9 ml final volume, mix 3900 µl CsTFA with 850 µl GB and adjust if the refractive index differs from nD-TC = 1.3702 +/- 0.0002. Again, it is advisable to prepare a slightly larger volume than needed.
  2. Add 3.59% vol. formamide (170 µl if mixed as above). Adjust if the refractive index differs from nD-TC = 1.3725 ± 0.0002. 
  3. Balance each tube pair.
  4. Centrifuge at 130,000 \(\times\) gav (42,400 rpm for the VTi 90 rotor) at 20 °C >65 h at maximum acceleration and minimum deceleration (no brake).

3.2 Gradient fractionation

  1. Stop the ultracentrifuge.
  2. Fill a 20-ml syringe with RNase-free water; remove any air bubbles.
  3. Attach the flexible tube to the syringe and mount it on the pump.
  4. Set the syringe pump to the desired speed. To collect 20 fractions, set the speed to 0.75 ml min-1 and collect in 20-second steps (make sure the correct syringe volume is also set).
  5. Connect a new 23G needle to the tube and test the flow. Wait until water starts to come out of the needle.
  6. Prepare 20, 1.5 ml non-stick tubes per gradient in a rack (assuming 20 fractions will be collected).
  7. Carefully remove the rotor from the centrifuge and release the screws or the lid. Ensuring that mechanical disturbance is minimal is crucial at this point.
  8. Mount 1 ultracentrifugation tube on the utility clamp about 1 cm above the opening of the first collection tube. 
  9. Carefully puncture the ultracentrifugation tube horizontally with the needle connected to the flexible tube, just below the bottom of the neck in the ultracentrifugation tube (the top level of the liquid volume). 
  10. Using a new 26G needle, carefully puncture a hole at the bottom of the ultracentrifugation tube and remove the needle. The ultracentrifugation tube should not leak at this stage.
  11. Place the rack under the ultracentrifugation tube so that the first collection tube is positioned right below the hole at the bottom of the tube.
  12. Start the pump and then start the stopwatch immediately after the first drop falls out of the ultracentrifugation tube.
  13. After 20 seconds, shift the rack so that the solution starts dropping to the second collection tube. Continue in a similar fashion until all tubes are filled.
  14. Discard the used ultracentrifugation tube and continue with the next gradient.
  15. After finishing fractionating all ultracentrifugation tubes, measure the density of every fraction using the refractometer starting from the last fraction (the lightest). The density of the fractions should increase at a linear rate.

3.3 Recovery of nucleic acids

3.3.1 DNA recovery

  1. To each 1.5 ml tube containing a gradient fraction, add 2 µl of GlycoBlue™ Coprecipitant (or 30 µg molecular-grade glycogen) (see Notes 4 and 5) and approximately 2 volumes of the PEG solution. Mix by inversion.
  2. Incubate the tubes for 2 h at RT.
  3. Centrifuge at > 13,000 \(\times\) g for 30 minutes at 4 °C. 
  4. Decant the supernatant, add 1 ml of 70% ethanol.
  5. Centrifuge for > 13,000 \(\times\) g for 10 minutes at 4 °C. 
  6. Decant the supernatant and leave the tubes open to dry at room temperature for ca. 15 min (preferably under an open flame or in a biological hood) to evaporate the remaining ethanol.
  7. Resuspend in 30 µl TE buffer or sterile water. Store at 4 °C up to several days or frozen at -20 °C or -80 °C indefinitely.
  8. Proceed with PCR amplification and sample preparation for sequencing using any standard protocol.

3.3.2 RNA recovery

  1. To each 1.5-ml tube containing a fraction, add 2 µl of GlycoBlue™ Coprecipitant (or 30 µg RNA-grade glycogen), 2.5 volumes of 100% ethanol and 0.1 volumes of sodium acetate (assuming 250-µl fractions were collected and 40 µl of each was used for density measurement, add 21 µl of Na-acetate and 625 µl of 100% ethanol). Mix by inversion.
  2. Incubate the tubes for 30 min at -80 °C.
  3. Centrifuge at > 13,000 \(\times\) g for 30 min at 4 °C.
  4. Decant the supernatant, add 1 ml of ice-cold 75% ethanol, invert the tube several times.
  5. Centrifuge at > 13,000 \(\times\) g for 15 min at 4 °C.
  6. Remove as much as possible from the supernatant first using a 1-ml tip, spin down the remaining drops in the tube, and remove the rest of the liquid with a 100-µl tip. Be careful not to disrupt the pellet.
  7. Leave tubes open to dry at room temperature for ca\(.~\)15 min (preferably under an open flame or in a biological hood) to evaporate the remaining ethanol.
  8. Resuspend the pellets in 10-µl RNase-free water or RNA Storage Solution. Proceed immediately to synthesising cDNA or store at -20 °C to -80 °C.
  9. Synthesise cDNA using any commercial reverse transcription kit (see Note 6).
  10. Proceed with PCR amplification and sample preparation for sequencing using any standard protocol.

4. Notes

  1. Substrate enrichment level. Typical SIP experiments involve using high substrate concentrations to achieve maximum labelling. Since 15N2 is also non-toxic, there is no limitation in supplying the incubation vials with atmospheric or even super-atmospheric concentrations of 15N2 gas (e.g., in anoxic incubations). However, this might not be necessary since even in very active systems only a small fraction of the dinitrogen gas eventually gets fixed. To save on costs, some of the gas can be replaced with another inert gas such as helium or argon. We have incubated several types of soil under an atmosphere of 40:40:20 (15N2, He, O2) and noticed no difference in labelling compared to incubating the samples under 80:20 (15N2, O2; data not shown), although this should probably be best confirmed for every type of sample.
  2.  Substrate contamination issues. Bottles of 15N2 are nearly always sold at a purity of around 99% (and >97% isotopic enrichment). However, the single remaining percent of foreign substance can turn out to be detrimental, because it was found out that a significant fraction of it is in the form of 15N-labelled ammonia and nitrate \cite{Dabundo_2014}. Ideally, every batch of labelled gas should be tested for potential contamination either by direct measurement of ammonia and nitrate, or, for example, indirectly by incubating a culture of a non-diazotrophic microorganism in the presence of the gas as a sole nitrogen source and then testing if the label has accumulated in the biomass. 
  3. Amount of template to use in a gradient. For DNA SIP, template amounts of 0.5--5 µg are typically used in the literature. There does not seem to be an upper limit to how much DNA can be loaded, but adding a large amount of an aqueous DNA solution will eventually significantly reduce the average density of the gradient.  The final amount of DNA that should be loaded on a gradient will depend on the size of the target guild compared to the total population and the downstream applications (i.e. PCR based analysis vs shotgun genomics). In contrast, much lower template amounts are used for  RNA SIP because it has been reported that RNA will precipitate in a CsTFA density gradient in concentrations above approx. 80 ng of RNA per ml of gradient solution \cite{Lueders_2003}. Luckily for targeting rRNA this is rarely an issue because rRNA accounts for over 80% of the total rRNA in a bacterial cell (with SSU rRNA alone accounting for ca. 27%), however for a transcriptomic analysis enrichment of mRNA might be needed \cite{Dumont_2011}.
  4. Type of glycogen. Standard molecular-grade (or RNA-grade) glycogen may also be used, but the pellet will most likely not be visible and can be accidentally lost in the washing process. Using dyed glycogen such as GlycoBlue™ helps to prevent loss of precipitated nucleic acids.
  5. Purity of glycogen. Non-molecular-grade glycogen may contain residual nucleic acids, and molecular-grade glycogen which is not RNA-grade may contain residual RNA. The presence of foreign DNA or RNA can significantly obscure the results of the SIP experiment and it therefore highly recommended to use an appropriate glycogen, and to verify by PCR that the glycogen is free of contaminating nucleic acids.
  6. cDNA synthesis. Because the RNA concentration in each fraction is very low (typically 1--300 ng, depending on how the gradient was designed and fractionated) the reverse transcriptase can be safely diluted 10-20 times before use without any noticeable effect on the reaction yield. Store at -20 to -80 °C.
Acknowledgements
The manuscript for this chapter was written online using authorea . RA was supported by BC CAS, ISB & SoWa RI (MEYS; projects LM2015075, EF16_013/0001782 – SoWa Ecosystems Research).
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