Sample Requirements

Illumina Input Recommendations

The Advanced Genomics Core prefers >200 ng for most assays. The higher input yields better performing assays and more robust data.

Illumina Assay Min Recommended Input Min Volume Quality and Concentration Minimums
Poly-A (standard) Library Prep 50 ng 25 µl RIN > 7 and 2 ng/µl
Poly-A (low input) Library Prep 1 ng 25 µl RIN > 7 and 0.05 ng/µl
Total RNA* (standard) Library Prep 10-40 ng 25 µl

DV200 >70%  – 0.80 ng/µl (10 ng total)

DV200 50-70% – 0.8 ng/µl (20 ng total)

DV200 30-50% – 1.6 ng/µl (40 ng total)

DV200 < 30% not recommended
Total RNA* (low input) Library Prep 1-4 ng 25 µl

DV200 >70%  – 0.04 ng/µl (1 ng total)

DV200 50-70% – 0.08 ng/µl (2 ng total)

DV200 30-50% – 0.16 ng/µl (4 ng total)

DV200 < 30% not recommended
small RNA Library Prep 10 ng 15 µl if submitting total RNA RIN > 7
DNA (standard) Library Prep 100 ng 25 µl 4 ng/µl
DNA (low input) Library Prep 0.5 ng 25 µl 0.02 ng/µl
User-made Library 10nM 25 µl < 1% adapter dimer

*The ribosomal depletion probes used during Total RNA library prep are selected based on the species indicated in the submission form. If both bacterial and human/mouse/rat probes are required, you must include both species in your form or indicate that it is mixed species. If your samples are isolated from blood and you require globin depletion, please indicate that in the submission notes.

Preferred Buffers:

  • RNA (DNase treated) – molecular biology grade water (NOT DEPC)
  • DNA – EB buffer (10mM TRIS (pH= 8.0-8.4)) or molecular biology grade water 
  • Illumina Libraries – EBT (10mM TRIS + 0.1%Tween20), EB (10mM TRIS)

Note: The presence of EDTA can inhibit enzymatic activity and lead to reduced final yield, failure of library prep, or low and uneven coverage when analyzed bioinformatically.

Oxford Nanopore Input Recommendations

Oxford Nanopore library preparation is sensitive to contaminants that can be revealed by Nanodrop readings. Note that the rapid chemistry is optimized for samples >30kb, while ligation sequencing can accommodate both HMW DNA and amplicons.

ONT Assay Recommended Input Min Concentration Min Volume Quality
Ligation Sequencing (with or without Native Barcoding) 1-2µg 25 ng/µl 30-50 µl 260/280 ~1.8-2.0
260/230 ~2.0-2.2
Rapid Barcoding Sequencing 500ng (≤12 samples)
100ng (>12 samples)		 55 ng/µl (≤12 samples)
6 ng/µl (>12 samples)		 10-15 µl 260/280 ~1.8-2.0
260/230 ~2.0-2.2

Preferred Buffers:

  • 10 mM TRIS (pH=8.0-8.4)

Note: We require that you provide us with a minimum 20ul aliquot of your elution buffer, so that we can use it to get accurate nanodrop readings when assessing the purity of your samples. This aliquot should be labeled with your Service Request ID and “EB”.

Other Assays Input Recommendations

Other Assay Min Recommended Input Min Volume
Quality Control NA 3-5 µl
BeadArray Genotyping 500 ng 10-15 µl
Single Cell/Single Nuclei (targeting 10K)
 40,000 40-50 µl

Preferred Buffers:

  • 10x Genomics – 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 μg/ml). If necessary, PBS can be replaced with most common cell culture buffers.

 

Note: Nanodrop can overestimate the amount of nucleic acid in a sample since it is not DNA/RNA-specific and can be influenced by the presence of contaminants. Fluorescence-based concentrations can be as much as 50% lower than those determined by nanodrop. If using nanodrop to quantify your samples prior to submitting to the AGC, be sure to at least double the amount of material being submitted.

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