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:

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.

Cycle determination for Ultralow Library Systems V2

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:

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:

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:

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

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:

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:

Library preparation

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:

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.