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Failure to add Partitioning Oil B to the row labeled 3 will prevent GEM generation and can damage the instrument. |
## g. Prepare for Run |
- l Close the lid (gasket already attached). DO NOT touch the smooth side of the gasket. DO NOT press down on the top of the gasket. Keep horizontal to avoid wetting the gasket. |
Run the chip in Chromium X series instrument immediately after loading the Partitioning Oil B. |
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60 μ l Master Mix + Cell Suspension in GEM-X 3' Chip |
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60 μ l Gel Beads in GEM-X 3' Chip |
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250 μ l Partitioning Oil B in GEM-X 3' Chip |
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GEM-X 3' Chip, closed |
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## 1.3 Run the Chromium X Series Instrument |
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Firmware Version 2.0.0 or higher is required if the Chromium X/iX instrument is used for this protocol. |
Firmware Version 2.2.0 or higher is required if the Chromium Xo instrument is used for this protocol. |
Consult the Chromium X Series Instrument User Guide with Readiness Test (CG000396) for detailed instrument operation instructions and follow the instrument touchscreen prompts for execution. |
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- a. Press the eject button on the instrument to eject the tray. If the eject button is not touched within 1 min , tray will close automatically. System requires a few seconds before the tray can be ejected again. |
- b. Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. Press the button to retract the tray. |
- c. Confirm GEM-X 3P program on the screen. Press the play button. |
- d. At completion of the run (~6 min) , the instrument will chime. Immediately proceed to the next step. |
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## 1.4 Transfer GEMs |
- a. Place a tube strip on ice. |
- b. Press the eject button of the Chromium X series instrument and remove the chip. |
- c. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. |
- d. Visually compare the remaining volume in rows labeled 1-2. Abnormally high volume in one well relative to other wells may indicate a clog. |
- e. Slowly aspirate 100 μl GEMs from the lowest points of the recovery wells in the top NO FILL row without creating a seal between the tips and the bottom of the wells. |
- f. Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels. Excess Partitioning Oil (clear) in the pipette tips indicates a potential clog. |
- g. Over the course of ~20 sec , dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes. |
- h. It is recommended to start GEM incubation within 5 min . If multiple chips are run back-to-back, place the GEMs from the subsequent chips in separate thermal cyclers. |
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## 1.5 GEM-RT Incubation |
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