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Sample requirements

The Otago Genomics & Bioinformatics Facility (OGBF) are now delivering services previously facilitated under the NZ Genomics collaboration directly to customers. Sequencing and bioinformatics enquiries can be directed to genomics@otago.ac.nz

RNA requirements

  • Intact Total RNA in nuclease-free water or elution buffer that does not contain EDTA (EDTA inhibits our library preparation).
  • Isolated or final purification by column/bead cleanup.
  • OD ratios: 260/280 >= 1.8; 260/230 >=1.5.
  • Agilent Bioanalyzer 2100 RIN >7 (negotiable, depending on species and source tissue).
  • If you cannot provide the amounts stated below, please contact us.

Sequencing

  • TruSeq Stranded mRNA library preparation input: 0.1-1 ug Total RNA in 50 uL, measured by qubit, the more the better to allow for internal QC.
  • TruSeq Stranded Total RNA library preparation input: 0.1-1 ug Total RNA in 10 uL, measured by qubit, the more the better to allow for internal QC.
  • Double-stranded cDNA for DNA-seq accepted by prior arrangement only.

Nanostring

Nanostring gene expression

  • 100 ng purified Total RNA  at >=20 ng/uL measured by qubit, the more the better to allow for internal QC.
  • The more intact the RNA the better, preferably with >=50% of fragments >300 nt.
  • Cell lysate is an option: 10,000 cells in 5 uL required; please contact us or Nanostring for specific requirements.

Nanostring miRNA

  • 100 ng purified intact Total RNA at >=33 ng/uL measured by qubit, the more the better to allow for internal QC.
  • For miRNA assays, 260/280 and 260/230 ratios must be >=1.8. Purity ratios <1.8 may not perform optimally, and will only be processed at your risk.
  • Plasma is an option for miRNA, but has specific requirements relating to blood collection and processing; discuss with us first.

Client RNA QC requirements prior to sample submission

  • A clear gel image and/or Bioanalyser trace of the total RNA so we can assess degradation. We require these to be sent with your sample submission form.
  • Details of the sample isolation method.
  • Quantification preferably by fluorometry, e.g Qubit, Ribogreen, otherwise spectrophotometry.*
  • OD260/280 ratio.
  • OD260/230 ratio.

* Quantification by fluorometry is critical, and is highly recommended: spectrophotometry often over-estimates concentration.

DNA requirements

  • Double-stranded, non-degraded (>20 kb with little smearing), preferably in EDTA-free Tris-based elution buffer, or nuclease-free water (EDTA may inhibit library preparation; if using TE, use a low EDTA formulation. Freeze samples to prevent degradation in the absence of a buffering agent).
  • Isolated by, or final purification with, column or bead cleanup.
  • OD260/280 ratio 1.8-2.0; 260/230 >1.5.
  • TruSeq Nano DNA library preparation: 300 ng (350 bp insert) at 10 ng/uL, minimum volume 30 uL; 500 ng (550 bp insert) at 10 ng/uL, min. vol. 50.
  • TruSeq PCR-free DNA library preparation: 1.5 ug (350 bp insert) or 2.5 ug (550 bp insert) at >=30 ng/uL, minimum volume 50 uL.
  • Low input (<10 ng) also an option, please contact us to discuss.
  • For other DNA-based library prep, e.g., ChIP-seq, exome capture, mitochondrial amplicons, or if you cannot provide the amounts/volumes stated above, please contact us for supported applications and input requirements.

Client DNA QC requirements prior to sample submission

  • A clear 0.6-0.8% agarose gel image in which (i) the sample has run well into the gel so we can assess degradation and RNA contamination; and (ii) includes a high MW ladder with a size range suitable for your organism (i.e., don’t use Kb+ or 100 bp ladder for mammalian and plant, use lambda HindIII, GeneRuler High Range Ladder, or equivalent).
  • Details of the sample isolation method.
  • Quantification preferably by fluorometry, e.g Qubit, Picogreen, otherwise spectrophotometry.*
  • OD260/280 ratio.
  • OD260/230 ratio.

* Quantification by fluorometry is critical and highly recommended: spectrophotometry often over-estimates concentration.