Question: What causes wetting failures?
Answer: A wetting failure occurs when GEM generation is impacted significantly, leading to improper emulsion formation and loss of single cell partitioning. Wetting failures have many causes (see below) but always result in a visibly heterogeneous emulsion with populations of very large GEMs. These can be detected visually in the pipette tip following emulsion retrieval (see figures below).
Causes:
1) 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 with cells and Gel Beads, air bubbles in the bottom of the Gel Bead or Master Mix well, loading less than the indicated volume of partitioning oil, or waiting too long after loading oil before running the chip).
2) 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.
3) 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.
4) 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).
5) Partial clogging due to sub-optimal 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.
6) Use of plasticware that is not recommended by 10x Genomics (e.g., pipette tips, strip tubes). This can can cause to various insults to proper GEM formation such as: shedding of fibers from the pipette tip filter, contaminating residual surfactant from the molding process, and/or errant plastic particles. For more details, please see: What is the importance of emulsion-safe plastic consumables?
7) Sometimes a wetting failure will occur despite proper loading of the chip. This happens occasionally but is a rare occurrence.
Recommendations:
1) If a wetting failure occurs during GEM generation, we recommend re-preparing the sample.
2) 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) a picture of the failed emulsions (in the pipette tips or the chip) to support@10xgenomics.com and (3) a recent purchase order and a contact phone number.
Best practices to minimize wetting failures:
- Add reagents in the stipulated order and wait 30 sec 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 min.
- Use only the recommended 10x plasticware. For more details, please see: What is the importance of emulsion-safe plastic consumables?
- 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 to a lab across the hall or up or down flights of stairs).
- Run the chip in the Chromium Controller 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.
- If using legacy chips (Chips A, B, E) please take note to follow the updated loading technique.
Figure 1. (From left) 1: Properly formed GEMs, 2: an example of a wetting failure in which the GEMs are not homogeneous, 3: properly formed GEMs, 4: another example of a wetting failure with complete separation of partitioning oil and reagents.
Figure 2. (From left) A: Properly formed GEMs, B: an example of a wetting failure in which the GEMs are not homogeneous, C: an example of a clog and wetting failure in which the GEMs are not homogeneous and a low emulsion volume is recovered.
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