virome-protocols

Table of contents

• Filtration
• Nucleic acid extraction
• DNase treatment
• SSIV split protocol (RT and 2nd strand synthesis)

Filtration

Material

• Filter (0.45 µm), TPP, 99745
• Syringe
• Container, 8 mL or 25 mL

Procedure

• Centrifuge sample and use supernatant to remove cells (e.g. WBC and RBC in case of blood) or other solids
• With syringe aspirate the sample and press through filter in a new container
• Replace filter when clogged

Nucleic acid extraction

Material

• 1 mL sample
• Pipettes and tips
• 1.5 mL tubes
• NucliSENS easyMag (bioMérieux)

Procedure

• from 1 mL sample extract total nucleic acid and elute in 25 µL using the NucliSENS easyMag
• store extract at -20°C or proceed immediately

DNase treatment

Reagents

• TURBO DNA-free Kit, Invitrogen, AM1907

Material

• DNA/RNA extract from nucleic acid extraction
• Pipettes and tips
• 1.5 mL tubes
• Thermomixer
• Centrifuge

Procedure

• Combine 10 µL DNA/RNA extract, 1.2 µL 10✕ TURBO DNase Buffer and 1 µL TURBO DNase (2 U)
• Incubate at 37°C for 20 min
• Add 2 µL resuspended DNase Inactivation Reagent
• Incubate 5 min at room temperature, mixing occasionally
• Centrifuge at 10'000 g for 1.5 min and transfer the RNA (supernatant) to a fresh tube

SSIV split protocol (RT and 2nd strand synthesis)

Reagents

• UltraPure DNase/RNase-Free Distilled Water, Invitrogen, 10977035
• Primer 6N, 100 µM (mh413_6N, 5'-NNNNNN-3')
• dNTP, Thermo Scientific, R0192
• SuperScript IV Reverse Transcriptase, Invitrogen, 18090200
• RNaseOUT Recombinant Ribonuclease Inhibitor, Invitrogen, 10777019
• RNase H, New England Biolabs, M0297S
• DNA Polymerase I, Large (Klenow) Fragment, New England Biolabs, M0210L
• Agencourt AMPure XP Beads, Beckman Coulter, A63881
• Ethanol absolute
• QuantiFluor ONE dsDNA System, Promega, E4870
• Nextera XT DNA Library Preparation Kit (96 samples), Illumina, FC-131-1096
• Nextera XT Index Kit (96 indexes, 384 samples), Illumina, FC-131-1002
• MiSeq Reagent Kit v3 (150-cycle), Illumina, MS-102-3001

Material

• DNA/RNA extract from nucleic acid extraction (for DNA workflow)
• DNase treated extract (for RNA workflow)
• Pipettes and tips
• 1.5 mL tubes
• PCR strips
• PCR cycler
• Magnetic Separation Rack
• Quantus Fluorometer, Promega, E6150
• Thermomixer
• MiSeq, Illumina

Procedure

• Fill in and follow instructions in SSIV_standard_split.xltx
• Purify total volume of 2nd strand synthesis products with 2x AMPure beads (20/30 µL DNA + 40/60 µL Beads, wash 2x with 80% Ethanol, dry Beads, elute DNA in 20 µL Water)
• Quantify DNA in 2nd strand synthesis products (Note: if unpurified, quantification might be too high)
• If purified 2nd strand synthesis products were below 0.2 ng/µL, 5 µL undiluted products were used for Nextera XT library preparation
• Follow Nextera XT standard protocol and sequence 151 cycles on the MiSeq

Release History

• 2.2.1
  • fix mistake, use 10 µL DNA/RNA extract for DNase treatment, not 5 µL
  • note that after extraction one could also proceed immediately
• 2.2.0
  • reduce ratio of AMPure beads from 3x to 2x for purification of 2nd strand synthesis products
• 2.1.1
  • describe centrifugation step before filtration
• 2.1.0
  • DNA/RNA split workflow including DNase treatment in RNA workflow
  • always purify 2nd strand synthesis products
• 2.0.0
  • no nuclease-treatment
  • switch RT enzyme from SSIII to SSIV
  • combined DNA/RNA workflow
  • exclude anker PCR
• 1.0.0
  • published virome-protocol, including DNA/RNA split workflow and anker PCR