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 .
