TITLE: Nucleic acids preparations
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 describes the nucleic acids preparations for the </span><span style = "font-style:italic;">Tara</span><span> Oceans expedition and is part of </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 class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/zhnfw26.jpg" /></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Figure 1</span><span>: Overview of -omics analysis strategy applied on Tara Oceans samples.</span></div></div></div>

[STEPS]
?. Please select nucleic acids extraction method.Euk_ DNA_RNA_extDNA/RNA extractions from size fractions 0.8–5 μm (or 0.8–3 μm), 0.8–2000 μm, 5–20 μm (or 3–20 μm), 3–2000 μm, 20–180 μm and 180–2000 μmDNA/RNA extractions from size fractions 0.2–1.6 μmgirus_DNA_extDNA extractions from sizData availability e fractions 0.2–1.6 μm, 0.1–0.2 μm, 0.45–0.8 μm, 0.2–0.45 μmvirus_DNA_extViral particle concentration and DNA extractions from size fraction
?. [Euk_ DNA_RNA_ext: filter disruption ]
Accommodate each filter into a grinding vial with 1 ml RA1 lysis buffer (included in the NucleoSpin RNA Midi kit) and 1% β-mercaptoethanol.
[RA1 lysis buffer]
[β-mercaptoethanol]
?. [Euk_ DNA_RNA_ext: filter disruption ]
Subject to the following grinding program using a 6,770 Freezer/Mill or 6,870 Freezer/Mill instrument :2 min pre-cooling timefirst grinding cycle at 10 knocks per second for 1 min1 min cooling timefinal grinding cycle at 10 knocks per second for 1 min.
?. [Euk_ DNA_RNA_ext: filter disruption ]
Resuspend the cryogrinded powder in 2 ml RA1 lysis buffer with 1% β-mercaptoethanol, transfer to a large capacity NucleoSpin Filter from RNA Midi kit and centrifuge for 10 min at 1,500 g.
[RA1 lysis buffer with 1% β-mercaptoethanol]
[Centrifugation]
?. [Euk_ DNA_RNA_ext: filter disruption ]
Add 1 ml RA1 lysis buffer with 1% β-mercaptoethanol.
[RA1 lysis buffer with 1% β-mercaptoethanol]
?. [Euk_ DNA_RNA_ext: filter disruption ]
Recentrifuge the filter for 3 min at 1,500 g.
[Centrifugation]
?. [Euk_ DNA_RNA_ext: filter disruption ]
Transfer the eluate to a new tube and add 1 volume of ethanol 70%. Mix well.
?. [Euk_ DNA_RNA_ext: DNA purification]
Elute DNA by three successive elutions each with 100 μl DNA elution buffer and store in sterile microtubes at −20 °C.
[Storage]
[DNA elution buffer]
?. [Euk_ DNA_RNA_ext: DNA quality control]
Quantify DNA by a dsDNA-specific fluorimetric quantitation method using Qubit 2.0 Fluorometer instrument with Qubit dsDNA BR (Broad range) and HS (High sensitivity) Assays.
?. [Euk_ DNA_RNA_ext: DNA quality control]
Optional: check DNA quality in a sample subset by running 1 μl on 0.7% agarose gel for 60 min at 100 V.
[Running 0.7% agarose gel]
?. [Euk_ DNA_RNA_ext: RNA purification]
Immediately continue RNA purification on the previous NucleoSpin RNA Mini spin column by digesting residual genomic DNA.Prepare DNase reaction mixture by add 10 μL reconstituted rDNase  to 90 μL Reaction Buffer for rDNase in a 1,5 ml tube. Mix by flicking the tube. Apply 95 μL DNase reaction mixture directly onto the center of the silica membrane of the column. Incubate at room temperature for 15 min.
[rDNase]
[Reaction buffer for rDNase]
?. [Euk_ DNA_RNA_ext: RNA purification]
Incubate column for 15 min at room temperature.
[Incubation]
?. [Euk_ DNA_RNA_ext: RNA purification]
Wash the column with RA2 and RA3 buffers according to the manufacturer's instructions.
?. [Euk_ DNA_RNA_ext: RNA purification]
Elute RNA in 60 μl RNase-free water and store in sterile microtubes at −80 °C.
[RNase-free water]
[Storage]
?. [Euk_ DNA_RNA_ext: RNA quality control]
Asses quantity of extracted RNA with RNA-specific fluorimetric quantitation on a Qubit 2.0 Fluorometer using Qubit RNA HS Assay.
?. [Euk_ DNA_RNA_ext: RNA quality control]
Assay quality of total RNA by capillary electrophoresis on a Bioanalyzer 2100 instrument (or equivalent), using the RNA 6,000 Pico LabChip kit.
?. [Euk_ DNA_RNA_ext: additional DNAse treatment on purified RNA]
In order to reduce as far as possible the risk of residual genomic DNA, apply a further DNase treatment.
The previous RNA extraction procedure includes an in-column DNase treatment. Based on previous experience with this method, this step is sometimes only partially effective and does not always preclude the presence of trace DNA in final RNA samples. DNA removal in RNA samples is essential to prevent the incorporation of any genomic material in the RNA-Seq library and consequently the misinterpretation of RNA-Seq data analyses.
?. [Euk_ DNA_RNA_ext: additional DNAse treatment on purified RNA]
Process the RNA samples further as follows: treat a quantity of 5 μg, or less, total RNA aliquots with Turbo DNA-free kit, according to the manufacturer’s DNase treatment protocol. Set up the reaction  by mixing total RNA with 5 µL 10X TURBO DNase Buffer and 1 µl DNAse in final 50 µl volume. Mix by gently flicking the tube. Do not vortex.
[Total RNA aliquots]
?. [Euk_ DNA_RNA_ext: additional DNAse treatment on purified RNA]
Incubate the reaction mixture for 30 min at 37 °C. (1/2)
[Incubation]
[Incubation]
?. [Euk_ DNA_RNA_ext: RNA purification]
Elute RNA in 9–15 μl nuclease-free water by two elution steps in order to maximize recovery.
[Nuclease-free water]
?. [Euk_ DNA_RNA_ext: RNA purification]
Purify the reaction mixture with the RNA Clean and Concentrator-5 kit following the procedure described for retention of >17 nt RNA fragments.
?. [Euk_ DNA_RNA_ext: additional DNAse treatment on purified RNA]
Add 2 µL DNAse, mix by gently flicking the tube (no vortex) and incubate the reaction mixture for 30 min at 37 °C. (2/2)
[Incubation]
[Incubation]
?. [Euk_ DNA_RNA_ext: RNA quality control]
Quantify purified RNA with Qubit RNA HS Assay.
The efficiency of DNase treatment  should  be assessed by PCR. When dealing with large number of samples, this validation step should be done on a subset of samples in order to check the efficiency of the treatment.
?. [DNA/RNA extractions from size fractions 0.2–1.6 μm]
Two different protocols are applied to these size fractions that mainly target prokaryotes. Viruses and giant viruses (giruses) are also recovered in these fractions although dedicated filters (e.g.,Please select from the following protocols: Acinas lab DNA extraction (Method ID: Acinas_prok_DNA_ext)Acinas lab RNA extraction (Method ID: Acinas_prok_RNA_ext)Genoscope lab DNA/RNA extraction (Method ID: Genoscope_prok_RNA_ext)
?. [Acinas_prok_RNA_ext]
RNA isolation is performed using the RNeasy Mini kit with a modified protocol.
?. [Acinas_prok_RNA_ext]
Cut the filters into small pieces and wash with pre-chilled PBS buffer in order to eliminate the RNA Later.
To avoid loss of any cell during the washing process, pass the washing solution through a glass cup filtration system including a filter of 0.22 μm pore-size.
?. [Acinas_prok_RNA_ext]
Place the pieces of the filter and the extra filter in a 50 ml falcon tube with a mixture of beads (1 ml of 0.5 mm glass beads and 2 ml of 0.1 mm Zirconia beads) and 10 ml buffer RLT+1% β-mercaptoethanol.
[0.5 mm glass beads]
[0.1 mm Zirconia beads]
[Buffer RLT+1% β-mercaptoethanol]
?. [Acinas_prok_RNA_ext]
Shake the mixture during 15 min in a MoBio Vortex (Vortex-Genie 2, MO BIO Laboratories, Inc.).
[Shaking]
?. [Acinas_prok_RNA_ext]
Centrifuge mixture at 4,500 g for 5 min at 4 °C.
[Centrifugation]
[Centrifugation]
?. [Acinas_prok_RNA_ext]
Transfer the supernatant to a new 50 ml falcon tube and centrifuge again for 10 min at 4,500 g.
[Centrifugation]
?. [Acinas_prok_RNA_ext]
Transfer the supernatant (about 10 ml) to a new 50 ml tube, add 1 volume 70% ethanol to the lysate and mix by inversion four times.
?. [Acinas_prok_RNA_ext]
Divide the mixture in two and load each volume separately (by successive 700 μl aliquots) in two RNeasy mini columns and filter using a vacuum pump.
In this way, a better yield is obtained rather than putting the entire volume in a single column.
?. [Acinas_prok_RNA_ext]
Wash each column with 700 μl Buffer RW1 and twice with 500 μl Buffer RPE according to the manufacturer protocol.
[Buffer RW1]
[Buffer RPE]
?. [Acinas_prok_RNA_ext]
Elute RNA from the membrane with 70 μl RNase-free water.
[RNase-free water]
The extraction protocol does not include a DNAse treatment. Perform a DNAse digestion, as described at the end of Euk_DNA_RNA_ext section prior to use for sequencing library preparation.
?. [Acinas_prok_RNA_ext: RNA quality control]
Quantify the RNA using both a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies Inc) and Qubit Fluorometer.
?. [Acinas_prok_DNA_ext]
Cut half of the 0.22 μm 142-mm-diameter Millipore polyethersulfone Express Plus membrane filter into small pieces and soak in 3 ml lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose).
[Lysis buffer]
?. [Genoscope_prok_RNA_ext]
Resuspend cryogrinded powders from each vial in 2 ml RA1 lysis buffer in the presence of 1% β-mercaptoethanol, then pool together and transfer to a single NucleoSpin Filter Midi.
[RA1 lysis buffer in the presence of 1% β-mercaptoethanol,]
?. [Genoscope_prok_RNA_ext]
Centrifuge for 20 min at 1,500 g.
[Centrifugation]
?. [Genoscope_prok_RNA_ext]
Add 1 ml RA1 lysis buffer with 1% β-mercaptoethanol to the column.
[RA1 lysis buffer with 1% β-mercaptoethanol]
?. [Genoscope_prok_RNA_ext]
Recentrifuge column for 3 min at 1,500 g.
[Centrifugation]
?. [Genoscope_prok_RNA_ext]
The RNA extraction procedure is integrated an in-column DNase treatment.
The previous RNA extraction procedure includes an in-column DNase treatment. Based on previous experience with this method, this step is sometimes only partially effective and does not always preclude the presence of trace DNA in final RNA samples. DNA removal in RNA samples is essential to prevent the incorporation of any genomic material in the RNA-Seq library and consequently the misinterpretation of RNA-Seq data analyses.
?. [Genoscope_prok_RNA_ext]
In order to reduce as far as possible the risk of residual genomic DNA, apply a further DNase treatment.
The previous RNA extraction procedure includes an in-column DNase treatment. Based on previous experience with this method, this step is sometimes only partially effective and does not always preclude the presence of trace DNA in final RNA samples. DNA removal in RNA samples is essential to prevent the incorporation of any genomic material in the RNA-Seq library and consequently the misinterpretation of RNA-Seq data analyses.
?. [Genoscope_prok_RNA_ext]
Process the RNA samples further as follows: treat a quantity of 5 μg, or less, total RNA aliquots with Turbo DNA-free kit, according to the manufacturer’s DNase treatment protocol.
[Total RNA aliquots]
?. [Genoscope_prok_RNA_ext]
Incubate the reaction mixture for 30 min at 37 °C. (1/2)
[Incubation]
[Incubation]
?. [Genoscope_prok_RNA_ext]
Elute RNA was in 9–15 μl nuclease-free water by two elution steps in order to maximize recovery.
[Nuclease-free water]
?. [Genoscope_prok_RNA_ext]
Purify the reaction mixture with the RNA Clean and Concentrator-5 kit following the procedure described for retention of >17 nt RNA fragments.
?. [Genoscope_prok_RNA_ext]
Incubate the reaction mixture for 30 min at 37 °C. (2/2)
[Incubation]
[Incubation]
?. [Genoscope_prok_RNA_ext]
Quantify purified RNA with Qubit RNA HS Assay.
The efficiency of DNase treatment  should  be assessed by PCR. When dealing with large number of samples, this validation step should be done on a subset of samples in order to check the efficiency of the treatment.
?. [girus_DNA_ext]
These size fractions are used to target giant viruses (giruses). DNA is extracted using a modified CTAB protocol.
?. [girus_DNA_ext]
Crush filters in liquid nitrogen, incubate at 60 °C for one hour in a CTAB buffer.
[Incubation]
[Incubation]
?. [girus_DNA_ext]
Purify DNA using an equal volume of chloroform/isoamyl alcohol (24:1) and a one-hour-long-RNase digestion step.
[RNase digestion]
?. [girus_DNA_ext]
Precipitate DNA with a 2/3 volume of isopropanol and wash with 1 ml of a solution containing 76% v/v ethanol and 10 mM ammonium acetate solution.
[Solution containing 76% v/v ethanol and 10 mM ammonium acetate solution]
?. [girus_DNA_ext]
Dissolve the extracted DNA samples in 100 μl of laboratory grade deionized water and stored at −20 °C until the sequencing steps.
[Laboratory grade deionized water]
[Storage]
?. [virus_DNA_ext]
Please follow steps of the following protocol: Preparation of Virus DNA from Seawater for Metagenomics
?. [Genoscope_prok_RNA_ext: filter disruption]
Cut the membranes in many small pieces, dispatch equally in two vials.
This procedure is based on cryogenic grinding followed by DNA/RNA purification with Nucleospin RNA kit as previously described for protists- and metazoans- enriched nucleic acids isolation (Euk_DNA_RNA_ext section)Modifications are applied in order to efficiently grind larger and thicker 0.2–1.6 μm and 0.2–3 μm filters.
?. [Genoscope_prok_RNA_ext: filter disruption]
Add 1 ml RA1 lysis buffer included in the NucleoSpin RNA Midi kit (Macherey-Nagel) and 1% β-mercaptoethanol (Sigma, St Louis, MO) to the grinding vials.
[RA1 lysis buffer]
?. [Genoscope_prok_RNA_ext: filter disruption]
Resuspend cryoground powders from each vial in 2 ml RA1 lysis buffer in the presence of 1% β-mercaptoethanol, then pool together and transfer to a single NucleoSpin Filter Midi.
[RA1 lysis buffer with 1% β-mercaptoethanol]
?. [Genoscope_prok_RNA_ext: filter disruption]
Centrifuge for 20 min at 1,500 g.
[centrifugation]
[Centrifugation]
?. [Genoscope_prok_RNA_ext: filter disruption]
Add 1 ml RA1 lysis buffer with 1% β-mercaptoethanol to the column.
[RA1 lysis buffer with 1% β-mercaptoethanol]
?. [Genoscope_prok_RNA_ext: filter disruption]
Recentrifuge column for 3 min at 1,500 g.
[Centrifugation]
Then, extraction was continued following the same procedure applied to protists- and metazoans- enriched samples, including the additional post-extraction DNase treatment, described in Euk_ DNA_RNA_ext section. (The steps from that protocol are also included below.)
?. [Genoscope_prok_RNA_ext: DNA purification]
Transfer the eluate to a new tube with addition of 1 volume of ethanol 70%.
?. [Genoscope_prok_RNA_ext: DNA purification]
Load the mixture to a NucleoSpin RNA Mini spin column and wash twice with 700 µl DNA wash solution.
?. [Genoscope_prok_RNA_ext: DNA purification]
Elute DNA by three successive elutions each with 100 μl DNA elution buffer and stored in sterile microtubes at −20 °C.
[Storage]
[DNA elution buffer]
?. [Genoscope_prok_RNA_ext]
Assess quantity and quality of extracted RNA with RNA-specific fluorimetric quantitation on a Qubit 2.0 Fluorometer using Qubit RNA HS Assay.
?. [Genoscope_prok_RNA_ext]
Quantify purified RNA with Qubit RNA HS Assay.
Initially, the efficiency of DNase treatment was assessed by PCR, showing that the treatment was efficient in all checked samples. Due to the large number of samples to be treated, this validation step was then omitted.
?. [Acinas_prok_DNA_ext]
Add lysozyme (1 mg ml−1 final concentration) and incubate samples at 37 °C for 45 min while slightly shaken.
[Incubation]
[Incubation]
?. [Acinas_prok_DNA_ext]
Add sodium dodecyl sulfate (1% final concentration) and proteinase K (0.2 mg ml−1 final concentration) and incubate samples at 55 °C for 60 min while slightly shaking.
[Incubation]
[Incubation]
?. [Acinas_prok_DNA_ext]
To collect the lysate and process with the standard phenol-chloroform extraction procedure, first, add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) to the lysate, carefully mix and centrifuge for 10 min at 3,000 g.
[Centrifugation]
?. [Acinas_prok_DNA_ext]
Recover the aqueous phase, add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) to the lysate, carefully mix and centrifuge for 10 min at 3,000 g.
[centrifugation]
?. [Acinas_prok_DNA_ext]
Add an equal volume of Chloroform:Isoamyl Alcohol (24:1, v/v) to the recovered aqueous phase in order to remove residual phenol.
?. [Acinas_prok_DNA_ext]
Centrifuge the mixture and recover the aqueous phase for further purification.
?. [Acinas_prok_DNA_ext]
Concentrate the aqueous phase and purify by centrifugation with a Centricon concentrator.
?. [Acinas_prok_DNA_ext]
Once the aqueous phase is concentrated, repeat this step three times adding 2 ml of sterile MilliQ water each time in order to purify the DNA.
[MilliQ water]
?. [Acinas_prok_DNA_ext]
After the third washing step, you should recover between 100 and 250 μl of purified total genomic DNA product per sample.
?. [Acinas_prok_DNA_ext]
Quantify isolated DNA using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA) and check its integrity on an agarose gel (1.5%).
?. [Acinas_prok_RNA_ext: RNA quality control]
Assay quality of total RNA by capillary electrophoresis on a Bioanalyzer 2100 instrument (or equivalent), using the RNA 6,000 Pico LabChip kit.
?. [Genoscope_prok_RNA_ext: DNA quality control]
Quantify DNA by a dsDNA-specific fluorimetric quantitation method using Qubit 2.0 Fluorometer instrument with Qubit dsDNA BR (Broad range) and HS (High sensitivity) Assays.
?. [Genoscope_prok_RNA_ext: DNA quality control]
Optional: check DNA quality in a sample subset by running 1 μl on 0.7% agarose gel for 60 min at 100 V.
[Running 0.7% agarose gel]
?. [Genoscope_prok_RNA_ext: RNA purification]
Immediately continue RNA purification on the previous NucleoSpin RNA Mini spin column by digesting residual genomic DNA.Prepare DNase reaction mixture by add 10 μL reconstituted rDNase  to 90 μL Reaction Buffer for rDNase in a 1,5 ml tube. Mix by flicking the tube. Apply 95 μL DNase reaction mixture directly onto the center of the silica membrane of the column.
[rDNase]
[Reaction buffer for rDNase]
?. [Genoscope_prok_RNA_ext: RNA purification]
Incubate column for 15 min at room temperature.
[Incubation]
?. [Genoscope_prok_RNA_ext: RNA purification]
Wash the column with RA2 and RA3 buffers according to the manufacturer's instructions.
?. [Genoscope_prok_RNA_ext: RNA purification]
Elute RNA in 60 μl RNase-free water and store in sterile microtubes at −80 °C.
[RNase-free water]
[Storage]
?. [Genoscope_prok_RNA_ext: RNA quality control]
Asses quantity of extracted RNA with RNA-specific fluorimetric quantitation on a Qubit 2.0 Fluorometer using Qubit RNA HS Assay.
?. [Genoscope_prok_RNA_ext: RNA quality control]
Assay quality of total RNA by capillary electrophoresis on a Bioanalyzer 2100 instrument (or equivalent), using the RNA 6,000 Pico LabChip kit.
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Process the RNA samples further as follows: treat a quantity of 5 μg, or less, total RNA aliquots with Turbo DNA-free kit, according to the manufacturer’s DNase treatment protocol. Set up the reaction  by mixing total RNA with 5 µL 10X TURBO DNase Buffer and 1 µl DNAse in final 50 µl volume. Mix by gently flicking the tube. Do not vortex.
[Total RNA aliquots]
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Incubate the reaction mixture for 30 min at 37 °C. (1/2)
[Incubation]
[Incubation]
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Elute RNA in 9–15 μl nuclease-free water by two elution steps in order to maximize recovery.
[Nuclease-free water]
?. [Genoscope_prok_RNA_ext: RNA purification]
Purify the reaction mixture with the RNA Clean and Concentrator-5 kit following the procedure described for retention of >17 nt RNA fragments.
?. [Euk_ DNA_RNA_ext: additional DNAse treatment on purified RNA]
Add 2 µL DNAse, mix by gently flicking the tube (no vortex) and incubate the reaction mixture for 30 min at 37 °C. (2/2)
[Incubation]
[Incubation]
?. [Euk_ DNA_RNA_ext: RNA quality control]
Quantify purified RNA with Qubit RNA HS Assay.
The efficiency of DNase treatment  should  be assessed by PCR. When dealing with large number of samples, this validation step should be done on a subset of samples in order to check the efficiency of the treatment.
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
In order to reduce as far as possible the risk of residual genomic DNA, apply a further DNase treatment.
The previous RNA extraction procedure includes an in-column DNase treatment. Based on previous experience with this method, this step is sometimes only partially effective and does not always preclude the presence of trace DNA in final RNA samples. DNA removal in RNA samples is essential to prevent the incorporation of any genomic material in the RNA-Seq library and consequently the misinterpretation of RNA-Seq data analyses.
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Process the RNA samples further as follows: treat a quantity of 5 μg, or less, total RNA aliquots with Turbo DNA-free kit, according to the manufacturer’s DNase treatment protocol. Set up the reaction  by mixing total RNA with 5 µL 10X TURBO DNase Buffer and 1 µl DNAse in final 50 µl volume. Mix by gently flicking the tube. Do not vortex.
[Total RNA aliquots]
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Incubate the reaction mixture for 30 min at 37 °C. (1/2)
[Incubation]
[Incubation]
?. [Genoscope_prok_RNA_ext: RNA purification]
Elute RNA in 9–15 μl nuclease-free water by two elution steps in order to maximize recovery.
[Nuclease-free water]
?. [Genoscope_prok_RNA_ext: RNA purification]
Purify the reaction mixture with the RNA Clean and Concentrator-5 kit following the procedure described for retention of >17 nt RNA fragments.
?. [Genoscope_prok_RNA_ext: additional DNAse treatment on purified RNA]
Add 2 µL DNAse, mix by gently flicking the tube (no vortex) and incubate the reaction mixture for 30 min at 37 °C. (2/2)
[Incubation]
[Incubation]
?. [Genoscope_prok_RNA_ext: RNA quality control]
Quantify purified RNA with Qubit RNA HS Assay.
The efficiency of DNase treatment  should  be assessed by PCR. When dealing with large number of samples, this validation step should be done on a subset of samples in order to check the efficiency of the treatment.