TITLE: DNA and RNA backups
AUTHORS: Adriana  Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of </span><a href="https://www.protocols.io/view/nucleic-acids-preparations-qwgdxbw?abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Nucleic acids preparations</span></a><span> for </span><a href="https://www.protocols.io/view/viral-to-metazoan-marine-plankton-nucleotide-seque-qv6dw9e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Viral to metazoan marine plankton nucleotide sequences from the </span><span style = ":;font-style:italic;">Tara</span><span style = ":;"> Oceans expedition</span></a><span>. </span></div></div>

[STEPS]
?. After nucleic acids extractions, prepare two RNA aliquots and three DNA aliquots for each sample. - RNA:  use one aliquot for the library preparation and sequencing process,  and store the second one as a backup. If RNA quantity is  - DNA: use the first aliquot for the librayr preparation and the second one for backup copy. The third aliquot is used to produce an amplified DNA backup by whole genome amplification (WGA) by using Illustra GenomiPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, UK) with the procedure described herein
?. Briefly, dilute 10 ng of DNA in 25 μl sample buffer and denature for 3 min at 95 °C.
[DNA]
[Sample buffer]
[Denaturation]
[Denaturation]
?. Cool samples on ice.
?. Mix samples to 22.5 μl reaction buffer containing random hexamers and 2.5 μl Phi29 enzyme mix and incubate at 30 °C for 3 hours.
[reaction buffer containing random hexamers and 2.5 μl Phi29 enzyme mix]
[Incubation]
[Incubation]
?. After amplification, heat inactivate Phi29 DNA polymerase during 10 min at 65 °C.
[Heat inactivation]
[Heat inactivation]
?. In order to reduce hyperbranched DNA regions generated by WGA process, incubate amplified DNA with RepliPhi phi29 DNA polymerase without any primer at 30 °C for 2 hours.
[Incubation]
[Incubation]
?. Digest  by S1 nuclease at 37 °C for 30 min.
[Incubation]
[Incubation]
?. Inactivate the enzyme at 65 °C for 3 min.
[Incubation]
[Incubation]
?. Purify DNA again with Agencourt GenFind V2 System and resuspend in 200 μl elution buffer.
[Elution buffer]
?. Quantify DNA with Qubit dsDNA BR and HS Assays and subject to quality check by running 1 μl on 0.7% agarose gel for 60 min at 100 V.
[DNA]
[Agarose gel]
?. [Clean up the reaction with Agencourt GenFind V2 System following the manufacturer protocol]
Omit the lysis step
?. [Repair internal nicks by adding 100 U E. coli DNA polymerase I in100 µl 1X NEB buffer 2 with 4 mM dNTP. Incubate 30 min at 25 °C]
?. [Repair internal nicks by adding 100 U E. coli DNA polymerase I in100 µl 1X NEB buffer 2 with 4 mM dNTP. Incubate 30 min at 25 °C]
25 °C
[incubation]
?. [Store DNA at -20 °C]