Jan 09, 2020

Public workspaceNucleic acids preparations

DOI
https://dx.doi.org/10.17504/protocols.io.qwgdxbw
Nucleic acids preparations
  • Adriana Alberti1,
  • Julie Poulain2,
  • Stefan Engelen2,
  • Karine Labadie2,
  • Sarah Romac3,4,
  • Isabel Ferrera5,
  • Guillaume Albini2,
  • Jean-Marc Aury2,
  • Caroline Belser2,
  • Alexis Bertrand2,
  • Corinne Cruaud2,
  • Corinne Da Silva2,
  • Carole Dossat2,
  • Frédéric vory2,
  • Shahinaz Gas2,
  • Julie Guy2,
  • Maud Haquelle2,
  • E'krame Jacoby2,
  • Olivier Jaillon2,6,7,
  • Arnaud Lemainque2,
  • Eric Pelletier2,
  • Gaëlle amson2,
  • Marc Wessner2,
  • Genoscope Technical Team2,
  • Silvia G. Acinas5,
  • Marta Royo-Llonch5,
  • Francisco M. Cornejo-Castillo5,
  • Ramiro Logares5,
  • Beatriz Fernández-Gómez5,8,9,
  • Chris Bowler10,
  • Guy Cochrane11,
  • Clara Amid11,
  • Petra Ten Hoopen11,
  • Colomban De Vargas3,4,
  • Nigel Grimsley12,13,
  • Elodie Desgranges12,13,
  • Stefanie Kandels-Lewis14,15,
  • Hiroyuki Ogata16,
  • Nicole Poulton17,
  • Michael E. Sieracki17,18,
  • Ramunas Stepanauskas17,
  • Matthew B. Sullivan19,20,
  • Jennifer R. Brum20,21,
  • Melissa B. Duhaime22,
  • Bonnie T. Poulos23,
  • Bonnie L. Hurwitz24,
  • Stéphane esant25,26,
  • Eric Karsenti10,14,27,
  • Patrick Wincker2,6,7
  • 1CEA, Institut de Biologie Intégrative de la Cellule;
  • 2CEA - Institut de Biologie François Jacob, Genoscope, Evry, France;
  • 3CNRS, UMR 7144, Station Biologique de Roscoff, France;
  • 4Sorbonne Universités, UPMC Univ Paris 06, UMR 7144, Station Biologique de Roscoff, France;
  • 5Departament de Biologia Marina i Oceanografia, Institute of Marine Sciences (ICM), CSIC, Barcelona, Spain;
  • 6CNRS, UMR 8030, Evry , France;
  • 7Université d'Evry, UMR 8030, Evry, France;
  • 8FONDAP Center for Genome Regulation, Santiago, Chile;
  • 9Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, El Libano Macul, Santiago, Chile;
  • 10Ecole Normale Supérieure, PSL Research University, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Paris, France;
  • 11European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genomes Campus, Hinxton, Cambridge , UK;
  • 12CNRS UMR 7232, BIOM, Banyuls-sur-Mer, France;
  • 13Sorbonne Universités Paris 06, OOB UPMC, Banyuls-sur-Mer , France;
  • 14Directors’ Research European Molecular Biology Laboratory, Heidelberg, Germany;
  • 15Structural and Computational Biology, European Molecular Biology Laboratory, Heidelberg, Germany;
  • 16Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, Japan;
  • 17Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA;
  • 18National Science Foundation, Arlington, Virginia, USA;
  • 19Departments of Microbiology and Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, Ohio, USA;
  • 20Department of Microbiology, The Ohio State University, Columbus, Ohio, USA;
  • 21Present address: Department of Oceanography and Coastal Sciences, Louisiana State University, Baton Rouge, Louisiana, USA;
  • 22Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan, USA;
  • 23University of Arizona, Tucson, Arizona, USA;
  • 24Department of Agricultural and Biosystems Engineering, University of Arizona, Tucson, Arizona, USA;
  • 25MARUM, Center for Marine Environmental Sciences, University of Bremen, Germany;
  • 26PANGAEA, Data Publisher for Earth and Environmental Science, University of Bremen, Germany;
  • 27Sorbonne Universités, UPMC Université Paris 06, CNRS, Laboratoire d’oceanographie de Villefranche (LOV), Observatoire Océanologique, Villefranche-sur-mer, France
  • Tara Oceans
Adriana Alberti
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DOI: https://dx.doi.org/10.17504/protocols.io.qwgdxbw
External link: https://www.nature.com/articles/sdata201793#methods
Protocol Citation: 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 vory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle amson, 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 esant, Eric Karsenti, Patrick Wincker 2020. Nucleic acids preparations. protocols.io https://dx.doi.org/10.17504/protocols.io.qwgdxbw
Manuscript citation:
Alberti, A. (2017). Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Scientific Data  4, 170093 (2017) doi: 10.1038/sdata.2017.93
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2018
Last Modified: January 09, 2020
Protocol Integer ID: 12968
Keywords: nucleic acids preparation, tara oceans sample, viral to metazoan marine plankton nucleotide sequence, nucleic acid, metazoan marine plankton nucleotide sequence, tara oceans expedition, oceans sample, oceans expedition, tara
Abstract
This protocol describes the nucleic acids preparations for the Tara Oceans expedition and is part of Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. 


Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.
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Guidelines
The 'Nucleic acids preparations' section describes different nucleic acids extraction methods applied to obtain DNA and RNA from the different plankton groups sampled during Tara Oceans Expedition.
1. DNA/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 μm (Method ID: Euk_ DNA_RNA_ext)
Plankton from these size fractions was collected on membrane filters and targeted unicellular eukaryotes (protists), usually <200 μm, and metazoans, usually >200 μm.
The protocol applied for nucleic acid extractions was based on simultaneous extraction of DNA and RNA by cryogenic grinding of cryopreserved membrane filters followed by nucleic acid extraction with NucleoSpin RNA kits (Macherey-Nagel, Düren, Germany) combined with DNA Elution buffer kit (Macherey-Nagel). This protocol was derived from optimization and validation experiments in the de Vargas laboratory at the Station Biologique de Roscoff (France). In particular, this preliminary work aimed principally to adapt the efficiency of the cell disruption and DNA/RNA extraction steps in order to efficiently capture nucleic acids from protists and metazoans collected from sea water filtering. Tests were conducted on a mock community composed of 26 monoclonal strains from the Roscoff Culture Collection (http://roscoff-culture-collection.org/) and natural filter samples collected in Roscoff (ASTAN, SOMLIT sampling). During these tests, the cell disruption step was optimized by applying a mechanical cryogrinding method to be sure that cells were efficiently disrupted, minimizing RNA and DNA degradation. Three cryogrinding protocols were tested using a 6,770 Freezer/Mill or 6,870 Freezer/Mill instrument (SPEX SamplePrep, Metuchen, NJ): 1 grinding cycle at 5 knocks per second for 1 min, 1 grinding cycle at 10 knocks per second for 1 min, and 2 grinding cycles at 10 knocks per second for 1 min. Best RNA and DNA quantities were obtained using 2 grinding cycles at 10 knocks per second for 1 min. Then, three simultaneous DNA /RNA extraction protocols were compared: Trizol method followed by RNeasy purification kit (Qiagen, Hilden, Germany), NucleoSpin RNA kit combined with DNA elution buffer set (Macherey-Nagel), and Nucleobond kit (Macherey-Nagel). Best quality results (DNA and RNA integrity conservation, ratios A260/280 and A260/230) were obtained with NucleoSpin RNA kit combined with DNA elution buffer set.
After validation, the procedure described herein was applied in the Genoscope laboratory on Tara Oceans filters from the size fractions cited above.
2. DNA/RNA extractions from size fractions 0.2–1.6 μm and 0.2–3 μm
Two different protocols were applied to these size fractions that mainly targeted prokaryotes. Viruses and giant viruses (giruses) were also recovered in these fractions although dedicated filters (e.g.,<0.22 μm) and specific extractions protocols (described in Sections 4 and 5) were allocated for their analysis.
3. DNA and RNA backups
After nucleic acids extractions, two RNA aliquots and three DNA aliquots were prepared for each sample. One aliquot was used for the library preparation and sequencing process, the second one was stored as a backup. The third DNA aliquot was used for a backup after a whole genome amplification (WGA) process described in the DNA and RNA backup section.
4. Viral particle concentration and DNA extractions from size fraction <0.22 μm (Method ID: virus_DNA_ext)
This protocol describes a technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer.
Briefly, FeCl3 precipitation was used to concentrate viruses from 20–60 l of 0.22 μm filtered seawater, which were then resuspended in ascorbate buffer (0.125 M Tris-base, 0.1 M sodium EDTA dehydrate, 0.2 M magnesium chloride hexahydrate, 0.2 M ascorbate). This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (>90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research. Data are also available from replicated metagenomes to help researchers’ decisions on the impact of linker amplification methods from low input DNA, viral purification strategies, and library preparation and sequencing platform choices. Following resuspension, recovered viruses were treated with DNase I to remove free DNA, followed by the addition of 0.1 M EDTA and 0.1 M EGTA to halt DNase activity, and further concentrated to<1 ml using an Amicon 100 KDa filter (Sigma). DNA was extracted using the Wizard Prep DNA Purification system (Promega, Madison, WI). DNA concentration was assessed with PicoGreen (Thermo Fisher Scientific). 
All detailed protocols are listed by name and are documented and available at https://www.protocols.io/groups/sullivan-lab.
5. DNA 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 μm (Method ID: girus_DNA_ext)
6. Preparation of single cell amplified genomes (SAGs) (Method ID: SAGs_amplif)
Single amplified genomes (SAGs) were generated and their taxonomic assignments were obtained as in Martinez-Garcia et al. with the following modifications. Samples for heterotrophic (aplastidic) cells were stained using SYBR Green I. Samples for phototrophic (plasidic) cells were unstained. No attempt was made to identify mixotrophic cells. Several 384-well plates containing single cells of each type were prepared from each environmental sample. Backup plates were stored frozen at −80 °C. Single cell amplifications were validated by using an aliquot for PCR with eukaryotic universal 18S primers. SAGs with positive 18S sequence were sent to Genoscope for whole genome sequencing. Upon arrival, 2.5 μl were removed from each well and used to generate an amplified DNA backup by WGA. The reactions were performed as described for DNA extractions (DNA and RNA backups) except that debranching reactions were omitted and instead amplified DNA was purified by QIAamp DNA Mini kit (Qiagen).
Table 1: Summary of libraries generated from Tara Oceans DNA and RNA samples and sequencing experiments performed on each type of library.


*Number of libraries with available readsets in public databases at the date of publication of the paper
Materials
STEP MATERIALS
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962.20
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962
Reagent70% Ethanol
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentRNeasy Mini KitQiagenCatalog #74104
ReagentPBS buffer
Reagent0.5 mm glass beads
Reagent0.1 mm Zirconia beadsBioSpec ProductsCatalog #11079101z
ReagentBuffer RLT 1% β-mercaptoethanol
Reagent70% Ethanol
Reagent142-mm-diameter Millipore polyethersulfone Express Plus membrane filterMerck Millipore (EMD Millipore)Catalog #GPWP14250
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentCTAB buffer
Reagentliquid nitrogen
ReagentChloroform
ReagentIsoamylalcohol
ReagentIsopropanol
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
ReagentEthanol 70%
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentLysozyme
ReagentSodium Dodecyl Sulfate
ReagentProteinase K
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
Reagentchloroform:isoamyl alcohol (24:1)
ReagentCentricon concentrator (Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane)Merck Millipore (EMD Millipore)
ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962.20
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962
Reagent70% Ethanol
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentRNeasy Mini KitQiagenCatalog #74104
ReagentPBS buffer
Reagent0.5 mm glass beads
Reagent0.1 mm Zirconia beadsBioSpec ProductsCatalog #11079101z
ReagentBuffer RLT 1% β-mercaptoethanol
Reagent70% Ethanol
Reagent142-mm-diameter Millipore polyethersulfone Express Plus membrane filterMerck Millipore (EMD Millipore)Catalog #GPWP14250
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentCTAB buffer
Reagentliquid nitrogen
ReagentChloroform
ReagentIsoamylalcohol
ReagentIsopropanol
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
ReagentEthanol 70%
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentLysozyme
ReagentSodium Dodecyl Sulfate
ReagentProteinase K
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
Reagentchloroform:isoamyl alcohol (24:1)
ReagentCentricon concentrator (Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane)Merck Millipore (EMD Millipore)
ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
Protocol materials
ReagentNucleoSpin RNA Midi Macherey-NagalCatalog #740962
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
ReagentRNeasy Mini KitQiagenCatalog #74104
Reagent0.5 mm glass beads
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
Reagent70% Ethanol
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
ReagentLysozyme
ReagentBuffer RLT 1% β-mercaptoethanol
ReagentIsoamylalcohol
ReagentProteinase K
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
ReagentCTAB buffer
Reagent142-mm-diameter Millipore polyethersulfone Express Plus membrane filterMerck Millipore (EMD Millipore)Catalog #GPWP14250
Reagentliquid nitrogen
ReagentSodium Dodecyl Sulfate
ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
ReagentPBS buffer
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962.20
ReagentEthanol 70%
ReagentChloroform
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
ReagentIsopropanol
ReagentCentricon concentrator (Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane)Merck Millipore (EMD Millipore)
Reagentchloroform:isoamyl alcohol (24:1)
Reagent0.1 mm Zirconia beadsBioSpec ProductsCatalog #11079101z
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962
ReagentPhenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)
Troubleshooting
Safety warnings
Please refer to the SDS (Safety Data Sheet) for warnings and hazard information.
Please select nucleic acids extraction method.
Euk_ DNA_RNA_ext
DNA/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 μm
DNA/RNA extractions from size fractions 0.2–1.6 μm
girus_DNA_ext
DNA 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 μm
virus_DNA_ext
Viral particle concentration and DNA extractions from size fraction <0.22 μm
Step case

Euk_ DNA_RNA_ext
22 steps

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.
Reagent1% β-mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
Amount1 µL RA1 lysis buffer
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962.20
Concentration1 Mass Percent β-mercaptoethanol
Subject to the following grinding program using a 6,770 Freezer/Mill or 6,870 Freezer/Mill instrument :
  • 2 min pre-cooling time
  • first grinding cycle at 10 knocks per second for 1 min
  • 1 min cooling time
  • final grinding cycle at 10 knocks per second for 1 min. 
Equipment
new equipment
NAME
6,770 Freezer/Mill or 6,870 Freezer/Mill instrumen
BRAND
(SPEX SamplePrep)
SKU
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.
Amount2 µL RA1 lysis buffer with 1% β-mercaptoethanol
Duration00:10:00 Centrifugation
ReagentNucleoSpin RNA MidiMacherey-NagalCatalog #740962
Add 1 ml RA1 lysis buffer with 1% β-mercaptoethanol.
Amount1 µL RA1 lysis buffer with 1% β-mercaptoethanol
Recentrifuge the filter for 3 min at 1,500 g.
Duration00:03:00 Centrifugation
Transfer the eluate to a new tube and add 1 volume of ethanol 70%. Mix well.
Reagent70% Ethanol
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.
ReagentNucleoSpin RNA/DNA Buffer SetMacherey-NagalCatalog #740944
Temperature-20 °C Storage
Amount100 µL DNA elution buffer
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.
Amount10 µL rDNase
Amount90 µL Reaction buffer for rDNase
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
Incubate column for 15 min at room temperature.
Duration00:15:00 Incubation
Wash the column with RA2 and RA3 buffers according to the manufacturer's instructions.
ReagentNucleoSpin RNAMacherey-NagalCatalog #740955
Elute RNA in 60 μl RNase-free water and store in sterile microtubes at −80 °C. 
Amount60 µL RNase-free water
Temperature-80 °C Storage
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. 
ReagentQubit dsDNA HS (High sensitivity) AssaysThermo Fisher ScientificCatalog #Q32851
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit dsDNA BR (Broad range) and HS (High sensitivity) Assays
SPECIFICATIONS
Optional: check DNA quality in a sample subset by running 1 μl on 0.7% agarose gel for 60 min at 100 V.
Duration01:00:00 Running 0.7% agarose gel
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. 
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS
Assay quality of total RNA by capillary electrophoresis on a Bioanalyzer 2100 instrument (or equivalent), using the RNA 6,000 Pico LabChip kit.
Equipment
new equipment
NAME
Bioanalyzer 2100 instrument
BRAND
G2939BA
SKU
with RNA 6,000 Pico LabChip kit
SPECIFICATIONS
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.
Note
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.
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.
Amount5 µL Total RNA aliquots
ReagentTurbo DNA-free KitInvitrogen - Thermo FisherCatalog #AM1907
Incubate the reaction mixture for 30 min at 37 °C. (1/2) 
Temperature37 °C Incubation
Duration00:30:00 Incubation
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) 
Temperature37 °C Incubation
Duration00:30:00 Incubation
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. 
ReagentRNA Clean & Concentrator-5 KitZymo ResearchCatalog #R1015
Elute RNA in 9–15 μl nuclease-free water by two elution steps in order to maximize recovery. 
Amount9 µL Nuclease-free water
Euk_ DNA_RNA_ext: RNA quality control
Quantify purified RNA with Qubit RNA HS Assay. 
Note
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.