Question: How do I diagnose and report a wetting failure with GEM-X assays?
Answer: When running GEM-X Chips, a wetting failure may occur. A wetting failure is evidenced by loss of proper emulsion formation and single cell partitioning. Wetting failures have many causes (see below) but always result in a visibly heterogeneous emulsion. Best practice is for recovered emulsions to be QC’d in pipette tips following emulsion retrieval (see figures below).
Causes:
- Incorrect priming of reagents in the microfluidic channels due to a chip loading error (e.g., not waiting for 30 seconds between adding the Master Mix and Gel Beads, loading less than the indicated volume of Partitioning Oil, or waiting too long after loading Gel Beads before loading Partitioning Oil).
- Presence of surfactant or other viscous reagents in the cell media/buffer that is added to the RT Master Mix. That can change the flow characteristics of the sample during partitioning.
- Running the instrument in an environment that is > 82°F or 27.7°C. Warmer temperatures will also change the way the fluids run through the chip.
- Reagents are at the wrong temperature (e.g., Gel Beads not equilibrated to room temperature immediately prior to chip loading, storage of Gel Beads at improper temperatures, or partial thaw and refreeze of Gel Beads).
- Partial clogging due to suboptimal quality of the biological sample. For example, excess extracellular debris can partially clog the microfluidic channels. Partial clogging can also be caused by poor cell health or compromised nuclei. Dying cells and compromised nuclei are both prone to clumping/aggregation; they have a propensity to leak chromatin and become sticky.
- Use of plasticware that is not recommended by 10x Genomics (e.g., pipette tips and strip tubes). This can cause various insults to proper GEM formation such as: shedding of fibers from the pipette tip filter, contaminating residual surfactant from the moulding process, and/or errant plastic particles. For more details, please see: What is the importance of emulsion-safe plastic consumables?
- Expired reagents. Using chips or reagents beyond their expiration date increases the risk of emulsion failures.
Recommendations:
- If a wetting failure occurs during GEM generation, we recommend re-preparing the sample.
Next steps:
- As part of our warranty policy, clog and wetting failures that have been properly documented are reimbursed with replacement reagents and chips.
- Please send (1) the lot numbers of the Gel Beads and chips that were used when the failure occurred, (2) date of the failure, (3) a picture of the failed emulsions in the pipette tips/strip tubes and the chip (see this article: How should I take photographs to document clogs and wetting failures?), and (4) a recent purchase order and a contact phone number to support@10xgenomics.com.
- Wetting failures are not eligible for replacement if expired reagents or chips were used.
- Wetting failures must be reported within 30 days of the expiration date, as stated in our Terms and Conditions.
- Wetting failures associated with an instrument error are only eligible for replacement if the instrument is under an active Warranty or Assurance Plan at the time of the failure.
Best practices:
- Do not use chips or reagents beyond their expiration date.
- Add reagents in the stipulated order and wait 30 seconds between the addition of Master Mix and the addition of Gel Beads. When loading more than one sample, load all samples first and then load the Gel Beads. We recommend not to extend the wait time between the addition of Master Mix and the addition of Gel Beads beyond 2 minutes.
- Use only the recommended 10x plasticware. For more details, please see: What is the importance of emulsion-safe plastic consumables?.
- After removing the chip from the sealed bag, use it within 24 hours.
- Avoid contacting the bottom surface of the chip with gloved hands and other surfaces. Frictional charging can lead to inadequate priming of the channels.
- Avoid introducing air bubbles when pipetting into the chip. If there is a bubble in the bottom of the well, you can carefully dislodge it so it can float to the top.
- After loading the chip, keep the chip level and avoid carrying the chip long distances (i.e., do not carry it to a lab across the hall or up or down flights of stairs).
- Troughs utilized for Partitioning Oil should be new and sterile.
- Run the chip in the Chromium X Series instrument immediately after the chip is loaded with reagents.
- Storage and handling of chip and instrument should be performed at temperatures < 82°F or 27.7°C.
- Take note to follow the updated loading technique required for GEM-X Chips.
The appearance of emulsions from the recovery wells of the GEM-X Chip after running the Chromium X Series instrument. The sample on the left is a successfully generated emulsion. The sample on the right has incomplete emulsion formation as there is separation of Partitioning Oil and reagents.
Very rarely, a wetting failure (A) may lead to minor changes in volumes recovered on other lanes of the GEM-X Chip (B-H), which does not impact data quality. The following may be observed with the other samples on the chip when a wetting failure occurs:
- Bubbles in the recovery wells
- ≥95 µl of emulsion volume recovered with air in the pipette tip(s)
Clogs are defined as recovering <100 µl GEMs which contain an excess of Partitioning Oil (See: How do I diagnose and report a clog failure with GEM-X assays?). Emulsions B-H do not contain an excess of Partitioning Oil and therefore, would not be eligible for replacements as they do meet the criteria of a “clog” with GEM-X Chips. We would recommend proceeding with the workflow with emulsions B-H.
Please note: lower emulsion volume recovered (<100 µl) may also be due to pipetting errors during chip loading (e.g., loading too little Master Mix volume for one or more samples) or recovering emulsions from the GEM-X Chip. As these emulsions are not clogs, they would not be eligible for warranty replacements. Be sure to use a well-calibrated multichannel pipette for chip loading and closely follow User Guide instructions for emulsion recovery.
Products: Single Cell Gene Expression, Single Cell Immune Profiling