Latest revision as of 22:58, 18 July 2022

scRNA-seq

(Continuing directly from Liberase dissociation or from FACS)

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.

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 = scRNA-seq =
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-<li>(Continue from the [[dissociation protocol]])</li>
+<li>(Continuing directly from [[LiberaseTM cell dissociation protocol|Liberase dissociation]] or from [[for FACS|FACS]])</li>
-*After 30um filter cup, immediately filter through 10um filter cup
+*Resuspend the cell pellet with 50-100uL AMEM(0) depending on the size of the pellet.
-Resuspend the cell pellet with 150-300uL AMEM(0) depending on the size of the pellet.
+*Count the cells using a hemocytometer.
-*Immediately sort the cells into 1.5mL tube with 700uL AMEM(0).
+**Standard protocol: 5uL cell solution + 5uL trypan blue, count the cells in 5 blocks
-**Normally we use F02 sorter because it has lasers for both GFP and mCherry.
+::: cell number/5 x 20 = cell number/uL
-*Note the FACS count.
+**Ideal concentration is between 800-1200 cells/uL
-*Centrifuge 300xrcf, 20 degree, 5 minutes. Remove supernatant.
+**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
-*The sample is ready for downstream applications:
+*Follow the 10X scRNA-seq protocol from here, or submit the samples to the NGS facility.
-:-> [[scRNA-seq]]
-:-> [[cell transplantation]]