Blue:RNA-Seq Experiments:07302013: Difference between revisions
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*Obtained PI stained nuclei from Gwen and Yun | *Obtained PI stained nuclei from Gwen and Yun | ||
*FACs sorted PI positive and FSC high population into 10ul FACS buffer in 96-well plate: | *FACs sorted PI positive and FSC high (i.e. largest) population into 10ul FACS buffer in 96-well plate: | ||
:#4000 nuclei - too low nuclei count | :#4000 nuclei - too low nuclei count | ||
:#6000 nuclei - too low nuclei count | :#6000 nuclei - too low nuclei count | ||
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:#20000 nuclei - 30,000/ml | :#20000 nuclei - 30,000/ml | ||
:#12000 nuclei in 2ul - 20,000/ml | :#12000 nuclei in 2ul - 20,000/ml | ||
*Used sort #3 - 120 nuclei/ul (6ul nuclei + 4ul C1 cell suspension solution - loaded 5ul) | *Used 10um beads on microscope - confirmed that nuclei are slightly larger than 10um (average of 12um) | ||
*Used sort #3 - 120 nuclei/ul (added 6ul nuclei + 4ul C1 cell suspension solution - loaded 5ul for approximately 360 nuclei total) | |||
*Captured approximately 1/4 of plate - several capture sites showed nuclei clumps - therefore there are issues with cell clumping and loss of sample after sorting... | *Captured approximately 1/4 of plate - several capture sites showed nuclei clumps - therefore there are issues with cell clumping and loss of sample after sorting... | ||
*Loaded and continued C1 reaction with captured wells | *Loaded and continued C1 reaction with captured wells |
Revision as of 00:45, 31 July 2013
Purpose
- Fluidigm C1 on human cadaver nuclei (J. Chun Lab)
Experiment
- Obtained PI stained nuclei from Gwen and Yun
- FACs sorted PI positive and FSC high (i.e. largest) population into 10ul FACS buffer in 96-well plate:
- 4000 nuclei - too low nuclei count
- 6000 nuclei - too low nuclei count
- 8000 nuclei - 120,000/ml
- 12000 nuclei - too low nuclei count
- 20000 nuclei - 30,000/ml
- 12000 nuclei in 2ul - 20,000/ml
- Used 10um beads on microscope - confirmed that nuclei are slightly larger than 10um (average of 12um)
- Used sort #3 - 120 nuclei/ul (added 6ul nuclei + 4ul C1 cell suspension solution - loaded 5ul for approximately 360 nuclei total)
- Captured approximately 1/4 of plate - several capture sites showed nuclei clumps - therefore there are issues with cell clumping and loss of sample after sorting...
- Loaded and continued C1 reaction with captured wells