Question: Should I proceed if there is an HT assay sample clog?
Answer: When running samples in the Chromium X, occasionally, a sample clog may occur. This is evidenced by recovering less than 180 µl GEMs from the recovery well (see figure below) or excess Partitioning Oil or air bubbles in the recovered GEMs. This is maybe visible in the first or second aspiration of 90 µl emulsion. If other samples are run in parallel, the clogged sample wells may have uneven volumes of remaining reagents compared to other sample wells.
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.
- We strongly recommend re-running 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. Please see HT-specific workflow instructions below.
- 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.
Workflow modification if proceeding with HT clog:
We strongly recommend rerunning the single cell suspension if a clog occurs. If the sample cannot be rerun, we have the following recommendations for processing HT sample clogs. This is an unsupported application-specific to HT-only, and cell recovery will be reduced. Only proceed with the following recommendations if the first emulsion aspirate is 90 µl.
- If the second emulsion aspirate is >60 µl, follow the protocol outlined in the User Guide as written.
- If the second aspiration is <60 µl, discard this emulsion and proceed with emulsion aspirate #1 only.
- GEM recovery and PCR cycles should not be adjusted.
- For cDNA pooling, in the absence of aspirate #2, proceed with 10 ul of cDNA and 10 µl EB. It may be possible to continue with more cDNA, but this is untested.
Best practices to minimize clogs:
To minimize Gel Bead clogging, follow these best practices:
- Make sure Gel Beads are completely thawed and vortex before 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 the likelihood of debris carryover to the sample line (increase cell concentration)
Figure 1. The appearance of GEMs from the recovery wells of the Single Cell HT chip after running the Chromium X. The three samples on the right are successfully generated emulsions. The sample on the left has an excess of Partitioning Oil (clear) and air in the pipette tip, indicating a clog.
Products: Single Cell Gene Expression, Single Cell Immune Profiling