Sample submission guide – bulk repertoire

Samples can be transferred to iRepertoire for library preparation and sequencing at any stage in the immune repertoire sequencing workflow from nucleic acid extraction to amplification to sequencing. Additionally, material can be sourced from a variety of different sample types. This page provides guidelines for sample submission whether you are planning to rely on iRepertoire for nucleic acid extraction, library preparation, sequencing, or all of the above.

Submitting samples for nucleic acid extraction

For submitting cells, we prefer submission of at least 1,000 isolated cells. However, because our technologies are sensitive enough to amplify down to the single cell level, we will accept as few as 250 cells. Researchers dealing with samples less than 250 cells will need to set up a project consultation. There are a few ways to store and send your cell samples, and your project needs will depend on which way is best for you.

  • If your samples do not need any downstream staining or sorting, we recommend storing them on QIAgen’s RNAprotect Cell Reagent. Spin down the cells, remove all supernatant, and completely re-suspend in RNAprotect Cell Reagent. Use volumes according to the manufacturer’s specifications. Cells that only need nucleic acid extraction can also be dry snap frozen, though extraction costs are higher than for samples stored in RNAprotect Cell Reagent.
  • If cells need to be sorted downstream (e.g., isolated T or B cell populations), we request cryopreserved cells at a concentration of 5 – 10*106 cells/mL of cryopreservation media with a viability (when thawed) of >70%. The recommended cryopreservation media is CryoStor CS5 or CS10 (BioLife Solutions).

To submit tissue, we can accept either FFPE samples or whole tissue biopies.

  • If you plan to submit FFPE samples, we request a minimum of 5 slides at 10 microns thick (or 50 microns worth of FFPE tissue). FFPE extraction is performed on a Covaris with acoustic sonication, which helps increase the yield of intact RNA.
  • We can also accept snap frozen tissue or tissue stored in RNAlater. These samples are outsourced for extraction to HudsonAlpha Discovery laboratory. The maximum tissue input for solid tissue is 30-40 mg per extraction.

For submitting whole blood, please see our extensive article on blood collection and sample considerations.  

Submitting genetic material for library preparation

For service samples, we request more nucleic acid than is required for a single reaction so that there is enough for a repeat amplification, should it be necessary. If the quantities listed below are difficult to obtain, we are experienced in achieving amplification from RNA input amounts as low as 200 pg. Quality and purity of the sample are much more important parameters than the input amount.

All samples should be submitted in nuclease free water and shipped in 1.5 mL microcentrifuge tubes. Please submit no less than 15 uL of sample for both RNA and DNA submissions.

For RNA we prefer quality to be either an A260/280 of ≥2.0 or a RIN value of 7 or higher. Our long read primer systems work best with RIN values higher than 7, but for samples with RIN values ranging from 5-7, we recommend our short read primers. We have also had success on samples with RIN values below 5.

  • FFPE RNA
    • minimum concentration of 5 ng/uL, minimum mass of 75 ng
    • optimum concentration of 20 ng/uL, optimum mass of 300 ng
  • Solid Tissue RNA
    • minimum concentration of 5 ng/uL, minimum mass of 75 ng
    • optimum concentration of 50 ng/uL, optimum mass of 1000 ng
  • PBMC/Whole Blood RNA
    • minimum concentration of 5 ng/uL, minimum mass of 75 ng
    • optimum concentration of 50 ng/uL, optimum mass of 1000 ng
  • Isolated Cell Populations
    • minimum concentration of 1 ng/uL, minimum mass of 15 ng
    • optimum concentration of 10 ng/uL, optimum mass of 200 ng

For gDNA, we generally require 1 – 5 µg per sample with an A260/280 ≥1.8 and a minimum concentration of 200 ng/µL. For gDNA extracted from more difficult sources such as FFPE, we typically request between 1-5 ug of gDNA at a minimum concentration of 70 ng/uL.

Suggestions for in-house library prep

The amount of RNA input you’ll want to use for library preparation with iRepertoire’s reagents is dependent on the starting sample type. The sample type influences the proportion of immune specific RNA in the prepared nucleic acid sample as well as the quality. Basically, the more purified your sample type, the less amount, in ng, of RNA template is required to achieve the desired immune-specific amplification.

If you are using total RNA from whole blood, tumors, or various other tissue types, and with no downstream cell sorting, then we recommend using 1 ug of RNA. There is likely a lot of non-immune specific RNA in these sample types that will act as background, so you want to make sure that you have enough immune specific RNA going into the reaction.

If you are using PBMCs, then you can use less RNA template, typically 300 – 500 ng. If you are using RNA extracted from a tissue type that has a lot of immune-specific cells, such as lymphoid tissue, then you can also get away with using less RNA template in your reaction.

When using cells that have been sorted, you can use even less RNA template because the concentration of immune-specific RNA is greater. When working with sorted cells, we have used as little as 20 pg (good quality, intact) RNA.

What also needs to be considered is the total volume that can be added to the first PCR reaction: 13.75 ul. The RNA needs to be at a concentration that will allow you to put the desired starting template amount (in ng) into this 13.75 ul. If you have a really concentrated RNA sample, then you can make up the difference of that 13.75 ul with nuclease-free water. For example, if your RNA sample is 250 ng/ul and you want to add 500 ng of template, you would add 2 ul of RNA template and 11.75 of water.

Ultimately, the appropriate amount of template added to the reaction must be determined empirically. If you are comparing one sample to another, researchers should begin with similar cell counts in addition to the same mass of template input per reaction.

In our experience, quality, NOT quantity, is the most important factor in a successful immune repertoire experiment. Planning the collection strategy, sample storage, and the nucleic acid extraction steps before a project starts pays dividends in terms of quality data once the actual lab-work begins.

Submitting prepared libraries for sequencing

We recommend that you send us 1ug of pooled library at a minimum concentration of 20ng/ul, and an A260/280 ratio of at least 1.8 (higher is fine). Pre-prepared libraries may be suspended in a wide variety of buffers, including 10 mM Tris, dH2O, or Qiagen EB. The most critical requirement is that buffers contain no more than 0.1 mM EDTA. If the concentration of the pooled libraries is too low, we recommend using a PCR purification kit to concentrate the sample by repeatedly applying the sample to the same column and eluting in a smaller volume.

Suggested Kits/Reagents

In general, commercial kits provide the best results, and we follow their protocols directly. We do recommend using an FFPE-specific kit for the FFPE samples, as these samples can be challenging. Further, we recommend using Quiagen kits over Beckman kits. In our hands, yield from the Beckman kits was​ significantly lower, around 20-30ng/ul for curls.

For nucleic acid extraction, we recommend the following kits:

  • RNA, Human and Mouse cells or tissue: Qiagen® RNeasy Mini kit  (www.qiagen.com catalog #74104). This kit is appropriate for cell counts between 5 x 105 – 1 x 107.
  • RNA, Human and Mouse cell samples with low counts: Qiagen® RNeasy Micro kit  (www.qiagen.com catalog #74004). This kit is appropriate for cell counts ≤ 5 x 105. This kit also contains Carrier RNA, which helps reduce the loss of sample RNA during the extraction process. Carrier RNA should be added prior to homogenization of cells when fewer than 500 cells are present.
  • RNA, Human and Mouse tissue:  Norgen Animal Tissue RNA Purification kit (www.norgenbiotek.com catalog #25700). We have tested this kit in the past with good results. If the tissue is not to be processed immediately, the Norgen kit recommends flash freezing in liquid nitrogen and storing at -70. If not flash-frozen, tissue samples should be stored in Qiagen® RNAlater (www.qiagen.com catalog #76106) at 2-8°C  for up to 4 weeks.
  • RNA, FFPE samples: Qiagen®  FFPE RNA (cat. #73504) purification kit. In house, we have also tested Stratagene’s Absolutely RNA FFPE kit (cat. no 400809) on lymph node slices and we know of other researchers who have used the kit on FFPE kidney biopsy samples. 
  • DNA and RNA, Human and Mouse cells or tissue: Qiagen® AllPrep DNA/RNA kits. These are available in Mini (catalog #80204), Micro(catalog #80284), and FFPE kits (catalog #80234).

For cells, samples to be used for submission or for subsequent RNA isolation should be resuspended in Qiagen® RNAprotect Cell Reagent (www.qiagen.com catalog #76526). Samples can be stored at 2-8°C for up to 1 week or -20°C indefinitely. For cryopreservation, our preferred media is CryoStor CS10, but we’ve received viable cell samples in good condition with homemade cryopreservation media as well.

For blood, please see our extensive article on blood collection and sample considerations.

General tips

Nucleic acids should be thawed on ice and handled in environments and reagents that are completely sterile and nuclease free. The environment surrounding the sample – including pipettes – can be cleaned with RNase Away or a similar cleaning reagent prior to extraction. All tubes and materials utilized should be certified RNase/Dnase free.

RNA is extremely sensitive to degradation. Ensure that RNA is stored at -80˚C prior to use and maintained at 4˚C during template addition steps. Gently flick RNA sample to mix once thawed, DO NOT VORTEX, and work quickly. Purified RNA may be stored in RNase-free water at -20°C for one month or -80°C for up to one year.  Under these conditions, no degradation of RNA should be detectable.  To avoid multiple freeze-thaws of RNA, we recommend aliquoting into smaller lots prior to freezing. Every freeze-thaw can damage the RNA with as much as a 20% reduction in clonotype discovery per thaw.  

Also, storing nucleic acid at higher concentrations can have a protective effect on the nucleic acid. To avoid template that is too dilute, we typically recommend using a commercially available RNA extraction column with an elution volume lower than the recommended. For example, if the protocol suggests elution in 50 µL, elute in 30-37 µL. If the RNA sample has already been extracted, iRepertoire can utilize our in-house concentration protocols to concentrate the RNA using a bead-based approach. However, reducing sample handling steps post-RNA extraction is advisable.

We do not recommend fixing samples. From our single cell experience, we have handled cells that were fixed with 1% PFA, and we have data indicating that the fixation does affect the downstream results. The successful single cell amplification was reduced by 60-70% compared to normal. In the bulk population, we would expect a similar effect. Even though we will still be able to obtain a library if enough cells are submitted, we know from our single cell experiments that the cross-linking of RNA affects the results.

Frequently asked questions

What is the minimum RNA Integrity Value (RIN) recommended?

In general, a RIN value >7 is recommended. However, we have found that our short read primers generally amplify samples with RIN>3, while our long read primers amplify samples with RIN>5.

Can RNA extracted from FFPE be used?

Yes, RNA from FFPE can be used. If the RNA is somewhat degraded (RIN value between 3 and 5), we recommend using our short read primers. If the RIN is >5, the long read primers should amplify the sample. For RIN values less than 3, the sample is likely too degraded to amplify.

Do you have a recommended RNA extraction kit for cells in suspension?

Yes, we recommend the Qiagen RNeasy Mini kit ( Cat. No. 74104) for cell counts above 5*10^5 (not to exceed 1*10^7 per column), and the Qiagen RNeasy Micro kit (Cat. No. 74004) for cell counts less than 5*10^5. For samples containing only a few hundred cells, we ask researchers to refer to our iPair service, as it might be a great option for the study and the extraction process can be skipped altogether.

Do you have a recommended kit for FFPE extraction?

Our preferred kit is Qiagen’s All-Prep (gDNA and RNA kit) for FFPE (cat. no. 80234). In house, we have also tested Stratagene’s Absolutely RNA FFPE kit (cat. no 400809) on lymph node slices with good success. We have also seen good results with the Stratagene kit from other researchers studying FFPE kidney biopsy samples. In general, commercial kits provide the best results, and we follow their protocols directly. We do recommend using an FFPE-specific kit for the FFPE samples, as these samples can be challenging.

Do you have a recommended kit for tissue RNA extraction?

We recommend the Norgen Animal Tissue RNA purification kit (cat. no. 25700). Typically, any commercial Tissue RNA extraction kit yields good results.

I have really low cell counts, should I use carrier RNA during RNA extraction?

Carrier RNA is typically recommended when you are extracting 500 cells or less. To prevent loss of RNA during extraction, carrier RNA should be added into the sample before RNA extraction process. If carrier RNA is used, an accurate RNA concentration of the original sample will be difficult to assess. Therefore, for samples with low cell counts, we recommend that you elute the RNA in a smaller volume to increase the eluted RNA concentration. For samples with extremely low cell counts, we ask researchers to refer to our iPair service. We can typically recommend a modification of this approach which will maximize the data obtained while bypassing the need for RNA extraction.