Difference between revisions of "For scRNA-seq"
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(Continuing directly from Liberase dissociation or from FACS)
(Created page with "= scRNA-seq = ---- <li>(Continue from the [[|dissociation protocol]])</li> *After 30um filter cup, immediately filter through 10um filter cup Resuspend the cell pellet with 15...") |
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= scRNA-seq = | = scRNA-seq = | ||
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− | <li>( | + | <li>(Continuing directly from [[LiberaseTM cell dissociation protocol|Liberase dissociation]] or from [[for FACS|FACS]])</li> |
− | * | + | *Resuspend the cell pellet with 50-100uL AMEM(0) depending on the size of the pellet. |
− | Resuspend the cell pellet with | + | *Count the cells using a hemocytometer. |
− | * | + | **Standard protocol: 5uL cell solution + 5uL trypan blue, count the cells in 5 blocks |
− | ** | + | ::: cell number/5 x 20 = cell number/uL |
− | * | + | **Ideal concentration is between 800-1200 cells/uL |
− | * | + | **If there are too many doublets or clumps, better repeat the experiment. |
− | + | **If the concentration is too low or too high -> spin down the cells and resuspend in a different volume | |
− | * | + | *Follow the 10X scRNA-seq protocol from here, or submit the samples to the NGS facility. |
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Latest revision as of 22:58, 18 July 2022
scRNA-seq
- Resuspend the cell pellet with 50-100uL AMEM(0) depending on the size of the pellet.
- Count the cells using a hemocytometer.
- Standard protocol: 5uL cell solution + 5uL trypan blue, count the cells in 5 blocks
- cell number/5 x 20 = cell number/uL
- Ideal concentration is between 800-1200 cells/uL
- If there are too many doublets or clumps, better repeat the experiment.
- If the concentration is too low or too high -> spin down the cells and resuspend in a different volume
- Follow the 10X scRNA-seq protocol from here, or submit the samples to the NGS facility.