Construction of shotgun libraries from RNA virus assemblages
Cesium chloride gradient is conducted to purify viruses from viral concentrate of seawater as described in others chapters of this book (Wommack et al. 2010, this volume; Steward and Culley 2010, this volume).
Each density fraction is then extracted with the QIAamp Viral RNA Mini kit (Qiagen) as directed by the manufacturer with the following exception (see annotation).
Remove contaminating DNA from RNA preparations with the Turbo DNAfree kit (Applied Biosystems) as described in the protocol provided with the kit.
In preparation for cDNA synthesis, 10 µL purified RNA viral template is mixed with a final dNTP concentration of 0.2 mM and 1 µM and 5 nM final concentrations of FR26RV-N and FR40RV-T primer, respectively.
The reaction is heated to 65°C then cooled on ice to allow the primers to anneal.
While still on ice, DTT (0.5 mM final conc.) is added to the reaction as an enzyme stabilization reagent with 40 U RNase OUT (Invitrogen) to protect the sample form RNAse activity.
The complementary DNA strand is synthesized with 200 U of Superscript III (Invitrogen) reverse transcriptase.
The reaction is incubated initially at 25°C for 10 min so that the hexamer 3′ end of primer FR26RV-N and the poly(T)20 3′ end of primer FR40RV-T remain annealed to the template while cDNA synthesis commences.
The temperature is then increased to 50°C, the temperature at which Superscript III’s processivity is highest, for 60 min.
After the hour-long incubation at 50°C, the first strand synthesis reaction is heated immediately to 94°C for 3 min and then rapidly cooled on ice.
A complementary second strand is subsequently synthesized at 37°C for 60 min with the addition of 2.5 U of Klenow Fragment, 3′-5′ exo – (New England Biolabs).
The Klenow reaction is terminated with a final incubation at 75°C for 10 min.
One PCR reaction contains 5 µL of template taken directly from the second strand synthesis reaction, 40 pM of FR20RV primer (see Table 1), a final dNTP concentration of 0.2 mM, 1 × Gold buffer, 2.5 mM MgCl2, and 2.5 U of Ampligold DNA polymerase (Applied Biosystems) in a final volume of 50 µL.
The reaction is incubated at 94°C for 10 min to fully denature the template and activate the hot start enzyme.
Followed by 35 cycles of denaturation at 94°C for 1 min.
Annealing at 65°C for 1 min.
Extension at 72°C for 2 min.
A final extension for 13 min that permits the completion of complementary strand synthesis.
Before gel separation, we purify and concentrate the PCR reactions with a MinElute PCR cleanup column (Qiagen) as described by the manufacturer.
Purified PCR products are loaded onto a 1% agarose gel containing 1 × SYBR safe stain (Invitrogen) and 0.5 × TBE buffer.
Bands of DNA of the appropriate size range are excised and purified with a MinElute Gel Extraction kit (Qiagen) according to the manufacturer’s instructions.
If the sample is to be cloned for Sanger sequencing, we recommend eluting DNA from the column with three washes of 10 µL nuclease-free water in preparation for the PCRTerminator (Lucigen) end repair reaction.
For the cloning protocol, please refer to the “Cloning and sequencing” section of "A degenerate primer reverse transcriptionpolymerase chain reaction–based protocol to determine the diversity of picorna-like viruses".
The relevant virus-containing gradient fractions are collected.
The CsCl in the Virus-containing gradient fractions are removed by buffer exchange using a centrifugal ultrafiltration unit with a nominal molecular weight cutoff of 30 KD to 100 KD (Microcon, Millipore or Nanosep, Pall Life Sciences) as described in Steward and Culley (2010).
Viruses are then recovered by eluting in 3 × 50 µL of 0.02-filtered SM buffer.
