Question: Should I proceed if there is a sample clog?
Answer: When running samples in the Chromium Controller, occasionally a sample clog may occur. This is evidenced by recovering less than 100 ul GEMs from the recovery well (see figure below) or an excess of Partitioning Oil in the recovered GEMs. If other samples are run in parallel, the clogged sample wells may have uneven volumes of remaining reagents compared to other sample wells.
Additional details can be found in our Technical Note:
Clogs are generally caused by sub-optimal sample preparations, non-sterile work environments, clumping of Gel Beads and/or slow chip loading.
Distinguishing between sample clogs and Gel Beads clogs:
- Gel Bead clogs are caused by improper handling of Gel Beads. A clog in the Gel Bead line causes Gel Beads to flow slower, leaving an excess of Gel Beads behind.
- Sample clogs are the result of suboptimal sample preparation of single-cell suspensions. They will result in faster than usual depletion of Gel Beads and lower cDNA yield.
To reduce the rate of microfluidic clogs, we recommend storing and loading chips in an area free of dust and debris. To further minimize the rate of microfluidic clogs resulting from the introduction of dust / debris during chip loading, pipette tip lids may be used to cover the open chip between loading steps. For HT chips (in which secondary holders are open while loading), a pipette tip lid may be placed over the chip itself and does not need to fully cover the opened portion with the gasket attached. Other materials other than pipette tip lids have not been tested, may introduce unwanted debris, and are not recommended.
We strongly recommend rerunning the sample if a clog occurs during GEM generation. However, if it is a precious sample in which additional cell material cannot be obtained and cells would be lost otherwise, it may be possible to continue with the protocol and generate cDNA, albeit at lower levels, and with the knowledge that the number of recovered cells will be lower. Specifically, if the clog occurred near the end of the run, most cells will have been partitioned into GEMs. Similarly, if a large number of cells have been run, this may also increase the likelihood of generating sufficient amounts of cDNA.
- 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 email@example.com, (3) a recent purchase order and a contact phone number.
Best practices to minimize clogs:
To minimize Gel Bead clogging follow these best practices:
- Make sure Gel Beads are completely thawed and vortex prior processing
- Handle Gel Beads in a sterile environment
- Store chips in an area free of dust
To minimize sample clogging follow these best practices:
- Dissociate cell clumps and aggregates and remove cell debris using cell strainers/filters; visually inspect single cell suspension before loading onto the chip
- Do not overload chip with excess cells
- Minimize the volume of single-cell suspension to reduce likelihood of debris carryover to sample line (increase cell concentration)
Figure 1. The appearance of GEMs from the recovery wells of the Single Cell chip after running the Chromium Controller. The sample on the left is a successfully generated emulsion. The sample on the right has an excess of Partitioning Oil (clear) and air in the pipette tip, indicating a clog. In some reagent clog cases, there is only 5-10 µl excess Partitioning Oil (and no air) in the pipette tip.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell ATAC