DNA-Seq systems
Sample isolation and input recommedations
- ds-cDNA (Rapid and Ultralow V2 only) or gDNA (all DNA-Seq systems)
- High quality, with an A260:A280 ratio of 1.8 or above
Note: While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples.
Fragmentation
For standard workflows, we recommend that cDNA or gDNA be fragmented using a Covaris Focused-ultrasonicator. Follow the settings for the desired fragment size for your specific ultrasonicator:
- DNA Shearing with M220
- DNA Shearing with S220 and E220
- DNA Shearing with S2 and E210
- DNA Shearing with LE220
Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® from New England BioLabs may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Technical Support for more information on these workflows.
Library preparation
For Ultralow V2, and Low Complexity Library Systems, we recommend performing a qPCR step with EvaGreen® Dye (Biotium) and the library amplification reagents in the kit prior to library amplification of any new sample or input into our kits to determine the optimal number of library amplification cycles.
Sequencing guidelines
NuGEN Ultralow and Low Complexity library systems are compatible with Illumina sequencing platforms.
Please note that low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.
RNA-Seq systems
cDNA generation (RNA-Seq Systems V2 and RNA-Seq FFPE Systems, Product nos. 7102,7150)
RNA input recommendations
- Total or poly A selected RNA
- High quality, with an A260:A280 ratio of 1.8 or above and a RIN score above 7
- DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include the Qiagen RNase-free DNase included with the RNeasy Mini RNA Purification and RNeasy Micro Purification Kits, or Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Columns. Note that column-based methods may not provide sufficient removal of DNA.
- While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
RNA isolation
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research QuickRNA TM Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
Ensure that the elution volume/final RNA concentration is appropriate for the cDNA kit used. NuGEN cDNA kits use input volumes of 5-10 uL.
Note: Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
cDNA clean-up
cDNA generated with the RNA-Seq V2 and RNA-Seq FFPE Systems should be cleaned up using a column-based method such as:
- QIAGEN MinElute Reaction Cleanup Kit
- QIAGEN QIAquick PCR Purification Kit
- Zymo Research DNA Clean & Concentrator TM -25
Alternatively, an SPRI bead-based system such as Agencourt® RNAClean® XP or AMPure XP Systems are suitable. If choosing a bead-based method, we recommend using a magnet compatible with 0.2 mL tubes, tube strips, or plates.
Note: Beads for final cDNA cleanup must be purchased separately from Beckman Coulter.
Library preparation
cDNA prepared by the RNA-Seq V2 and FFPE RNA-Seq Systems are compatible with any library preparation kit that accepts ds-cDNA as input.
Note: cDNA generated by the RNA-Seq System V2 must be fragmented prior to library prep.
We recommend use of NuGEN’s Ultralow V2 or Rapid library systems.
RNA-Seq library systems (Universal RNA-Seq, Complete Prokaryotic RNA-Seq, and SoLo RNA-Seq Systems)
RNA input recommendations
- Total or poly A selected RNA (Universal and Complete Prokaryotic RNA-Seq Systems)
- RNA from exosomes, cfRNA, nuclear RNA, or whole cell inputs (SoLo only)
- High quality, with an A260:A280 ratio of 1.8 or above and a RIN score above 7
Note: While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
- DNase treated (Universal RNA-Seq systems, Complete Prokaryotic systems). We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include the Qiagen RNase-free DNase included with the RNeasy Mini RNA Purification and RNeasy Micro Purification Kits, or Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Columns. Note that column-based methods may not provide sufficient removal of DNA.
RNA isolation
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research QuickRNA TM Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
Note: Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
Fragmentation (optional)
- For standard workflows, we recommend that cDNA be fragmented to an average of 200 bp using a Covaris Focused-ultrasonicator.
- Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® from New England BioLabs may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Technical Support for more information on these workflows.
Library preparation
We recommend performing a qPCR step with EvaGreen® Dye (Biotium) and the library amplification reagents in the kit prior to library amplification of any new sample or input into our kits to determine the optimal number of library amplification cycles.
- Cycle determination for Universal library systems
- Cycle determination for Single Cell and SoLo library systems
Library preparation
We recommend performing a qPCR step with EvaGreen® Dye (Biotium) and the library amplification reagents in the kit prior to library amplification of any new sample or input into our kits to determine the optimal number of library amplification cycles.
- Cycle determination for Universal library systems
- Cycle determination for Single Cell and SoLo library systems
Sequencing guidelines
NuGEN RNA-Seq library systems are compatible with Illumina sequencing platforms.
Please note that low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.
Target Enrichment systems
RNA workflows
RNA input recommendations
- Total RNA
- High quality, with an A260:A280 ratio of 1.8 or above and a RIN score above 7
Note: While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
- DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include the Qiagen RNase-free DNase included with the RNeasy Mini RNA Purification and RNeasy Micro Purification Kits, or Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Columns. Note that column-based methods may not provide sufficient removal of DNA.
RNA isolation
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research QuickRNA TM Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
Note: Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
Fragmentation
Fragmentation of cDNA generated for Fusion Panel Target Enrichment is not recommended.
DNA workflows
Sample isolation and input recommendations
- gDNA inputs
- High quality, with an A260:A280 ratio of 1.8 or above
Note: While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples.
Fragmentation
For standard workflows, we recommend that cDNA or gDNA be fragmented using a Covaris Focused-ultrasonicator. Follow the settings for the desired fragment size for your specific ultrasonicator:
- DNA Shearing with M220
- DNA Shearing with S220 and E220
- DNA Shearing with S2 and E210
- DNA Shearing with LE220
Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® from New England BioLabs may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Technical Support for more information on these workflows.
Library preparation
We recommend performing a qPCR step with EvaGreen® Dye (Biotium) and the library amplification reagents in the kit prior to library amplification of any new sample or input into our kits to determine the optimal number of library amplification cycles.
Sequencing guidelines
NuGEN Target Enrichment library systems are compatible with Illumina sequencing platforms.
Please note that low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.
Methyl-Seq systems
Sample isolation and input recommendations
- gDNA
- High quality, with an A260:A280 ratio of 1.8 or above
Note: While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples.
Fragmentation
For standard workflows, we recommend that cDNA or gDNA be fragmented using a Covaris Focused-ultrasonicator. Follow the settings for the desired fragment size for your specific ultrasonicator:
- DNA Shearing with M220
- DNA Shearing with S220 and E220
- DNA Shearing with S2 and E210
- DNA Shearing with LE220
Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® from New England BioLabs may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Technical Support for more information on these workflows.
Library preparation
NuGEN Ultralow Methyl-Seq and RRBS Methyl-Seq library systems require bisulfite conversion of DNA for successful library preparation. We recommend using the EpiTect Fast Bisulfite Conversion Kit from Qiagen. While we have not tested alternative workflows, other bisulfite conversion kits may be acceptable.
Sequencing guidelines
NuGEN Ultralow Methyl-Seq and RRBS Methyl-Seq library systems are compatible with Illumina sequencing platforms. A custom sequencing primer, MetSeq1, is required for the forward read and is provided with the kit.
Please note that low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.