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b. Add 28.4 μl Master Mix into each tube of a PCR 8-tube strip on ice. |
| Master Mix Add reagents in the order listed | Master Mix Add reagents in the order listed | PN | 1X ( μ l) | 4X + 10% ( μ l) | 8X + 10% ( μ l) | |
|-----------------------------------------------|-----------------------------------------------|-----------------|-------------|-------------------|-------------------| |
| | RT Reagent E | 2001106 | 16.3 | 71.7 | 143.4 | |
| | Template Switch Oligo B | 2001027 | 3.1 | 13.6 | 27.3 | |
| | Reducing Agent B | 2000087 | 2.0 | 8.8 | 17.6 | |
| | RT Enzyme E | 2001105/2001146 | 7.0 | 30.8 | 61.6 | |
| | Total | - | 28.4 | 125.0 | 249.9 | |
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## Assemble GEM-X Chip |
The GEM-X chip is only compatible with Chromium X/iX Chip Holder (also referred to as Chromium X Series Chip Holder; PN-3000598). DO NOT use any other holder. |
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See Tips & Best Practices on page 18 for chip handling instructions. |
- l Close the holder lid. |
- l Attach the gasket by holding the tongue (curved end, to the right) and hook the gasket on the left-hand tabs of the holder. Gently pull the gasket toward the right and hook it on the two right-hand tabs. |
- l DO NOT touch the smooth side of the gasket. |
- l Open the chip holder. |
- l Remove the chip from the sealed bag. Use the chip within ≤ 24 h . |
- l Align notch on the chip (upper left corner) and the open holder with the gasket attached. |
- l Slide the chip to the left until the guide on the holder is inserted into the chip. Depress the right hand side of the chip until the spring-loaded clip engages. |
A generic image representative of the GEM-X 3' Chip is shown on the next page. |
- l Keep the assembled unit with the attached gasket open until ready for and while dispensing reagents into the wells. |
- l After loading reagents, close the chip holder. DO NOT press down on the top of the gasket. |
For GEM generation, load the indicated reagents only in the specified rows, starting from row labeled 1, followed by rows labeled 2 and 3. DO NOT load reagents in the top row labeled NO FILL. See step 1.2 for details. |
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## Cell Suspension Volume Calculator Table |
For step 1.2 of this protocol. |
DO NOT add PBS directly to single cell suspension. Add PBS to the Master Mix. See step 1.2c. |
Cell Recovery Target - 500-10,000 |
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## Cell Recovery Target - 10,000-20,000 |
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