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Sample Submissions

Submitting samples? Check out our guidelines to ensure your project is delivered safe an sound. Start success here!

Library

Library may be submitted as individual samples or as pooled library. Individual samples should be at least 7nM in 15uL due to QC measures needed before pooling. Please let us know if you are submitting individual libraries that do not meet this requirement.

Pooling Recommendations

An Adept reaction processes one linear library or a pool of ≤ 384 indexed linear libraries. When pooling, uniquely index each library in the pool and apply the following criteria to pool libraries with similar characteristics:

• Pool libraries that require the same run parameters.

• Do not pool Adept libraries with Elevate libraries.

• Balance the concentrations of libraries in a pool based on the sequencing throughput requirements for each sample. To maintain balance after library prep, make sure the libraries have similar size distributions.

Library Amount

The final QC+circularization protocol requires which is equivalent to ≥ 6.67--16.67 nM pooled library in 40uL of 10 mM Tris buffer pH 7.5--8.5 or similar solution. The concentration of EDTA in the library CANNOT exceed 1 mM. Linear library has high stability at room temperature, though it is recommended that during shipping, delivery, and submission, samples remain on ice.

**Update: We are now allowing submission of libraries at 3nM in 26uL**

Fragment Size
Ideal library size is between 250-800bp. Library portions that contain > 1000 bp sequences may impact Q30 scores and require adjustment for density. Avoid significant amounts of adapter dimer or other short byproducts (< 175 bp). If the input library contains short byproducts, AUGenomics recommends additional cleanup using sample purification beads. 

Diversity Requirement

Please see PhiX recommendations below and note necessary PhiX on your Submission Form.

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Image by julien Tromeur

Extracted Material

Extracted material may be submitted for library preparation services. Please ensure best practices are followed during your extraction protocols to prevent degradation or impurities in your sample. Elution buffers may be used, though RNAse/DNAse free water is an acceptable standard. Do not suspend samples in buffer containing EDTA, it will inhibit downstream reactions

RNA

Please follow best practices when working with RNA to eliminate potential degradation. The RNA sample should be free of salts (e.g., Mg2+ and guanidinium salts), divalent cation chelating agents (e.g., EDTA and EGTA) or organics (e.g., phenol and ethanol). RNA must be free of DNA. gDNA is a common contaminant from RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I to remove all traces of DNA (DNase is not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of the DNase I may degrade the oligos necessary for the enrichment.

Library Amount

Samples should be suspended in nuclease-free water. For most preps,  high quality RNA with a RIN score > 7 is required. Please ensure extracted RNA are shipped on dry ice for overnight delivery. 

 

Poly(A)/mRNA

  • RIN > 7

  • 200 pg/µL in 60µL (For higher rate of PCR duplicates)

  • 20 ng/µL in 60µL (For lowest rate of PCR duplicates)

Total RNA/ Whole Transcriptome

  • RIN > 7 or partially degraded RNA samples (RIN = 2 to 7)

  • 5 ng/µL in 15µL (For higher rate of PCR duplicates)

  • 90 ng/µL in 15µL (For lowest rate of PCR duplicates)

 

Immune Repertoire

Enriched B cell or T Cell or B cell and T Cell total RNA from peripheral blood mononuclear cells (PBMCs). 

  • RIN > 7

  • 5 ng/µL in 15µL (For higher rate of PCR duplicates)

  • 90 ng/µL in 15µL (For lowest rate of PCR duplicates)

Low Input RNA

  • RIN > 8

  • .5 pg/µL in 15µL (For higher rate of PCR duplicates)

  • 50 pg/µL in 15µL (For lowest rate of PCR duplicates)

 

Small RNA

Small RNA fragments should have a 5´ phosphate and 3´ OH to ligate and must be free of ATP.

  • RIN > 7 

  • 17 ng/µL in 11µL (For higher rate of PCR duplicates)

  • 170 ng/µL in 11µL (For lowest rate of PCR duplicates)

DNA

It is important to remove all cations and chelators from DNA preparations. Make sure input DNA is in water, 10 mM Tris-HCl (pH 8.0), or buffer EB.

 

Standard DNA

  • DIN > 7 

  • 1 ng/µL in 40µL (For higher rate of PCR duplicates)

  • 100 ng/µL in 40µL (For lowest rate of PCR duplicates)

Exome

  • DIN > 8

  • 10 ng/µL in 15µL (For higher rate of PCR duplicates)

  • 100 ng/µL in 15µL (For lowest rate of PCR duplicates)

Image by National Cancer Institute

Cells

We recommend between 1x10^5 and 5x10^6 cells for submission. Although cell pelleting is preferred, we may accept alternative cell submissions. Some alternatives, such as in sub-recommended cell count situations, require a pre-made master mix to sort into. Please contact us when planning an alternative method.

Fresh

Please book a submission appointment before submitting Fresh cells.

Snap Frozen

Cell pellets may be sent snap frozen and submitted on dry ice. To snap freeze, place the cells in a tube for long term storage (cryotube or Eppendorf tube with parafilm wrapped around the top) and either submerge the tube in liquid nitrogen or a liquid-nitrogen cooled bath (e.g., isopentane) or place the tube deep in a bucket of dry ice.

Trizol or equivalent lysis

Ensure that 1mL Trizol Reagent is added per 5x10^6 cells in pellet, homogenize sufficiently, then freeze. Samples in Trizol must be delivered on dry ice.

Image by National Cancer Institute
Image by Piron Guillaume
Image by Piron Guillaume

Tissue

Tissue may be submitted as either Fresh, Snap Frozen, in lysis reagent, or FFPE. Although a minimum of 3mg tissue is required, approximately 10mg is generally recommended for most tissue submissions.

Fresh

Allprotect® Tissue Reagent by Qiagen or similar is required for submitting fresh tissue to stabilize DNA, RNA, and proteins. At least 10 volumes of Allprotect (or approximately 100 ul reagent per 10 mg of tissue) are required. After fully submerging samples, samples are stable up to 37C for 24h, 15--25C for 7 days, or 2--8C for 6 months. Allprotect Tissue Reagent is intended for use with tissues only, and is not suitable for stabilizing DNA, RNA, and proteins in cultured cells, whole blood, plasma, or serum. Please read AllProtect handbook before first-time use.

Snap Frozen

Tissue may be sent snap frozen and submitted on dry ice. To snap freeze, place the tissue in a tube for long term storage (cryotube or Eppendorf tube with parafilm wrapped around the top) and either submerge the tube in liquid nitrogen or a liquid-nitrogen cooled bath (e.g., isopentane) or place the tube deep in a bucket of dry ice.

Trizol or equivalent

All tissues submitted in Trizol must be adequately homogenized before hand-off or storage. Ensure that 10 volumes of Trizol are added to sample (1mL Trizol per 50--100mg). Samples in Trizol must be delivered on dry ice. 

FFPE block

Standard formalin-fixation and paraffin-embedding procedures result in significant fragmentation of nucleic acids. To limit the extent of DNA fragmentation, be sure to fix tissue samples in 4-10% formalin as quickly as possible after surgical removal. Use a fixation time of 14-24 hours (longer fixation times lead to more severe DNA fragmentation, resulting in poor performance in downstream assays).

Thoroughly dehydrate samples prior to embedding. Store and deliver FFPE samples at low temperatures (2--8C). Four sections of 10--20um thickness should be available to cut. 

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