Blue:RNA-Seq Experiments:11092016
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Kidney Samples: Nuclei isolation for Drop-seq[edit]
- Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative:
- Isolated Nephrons (intact X8; see attached photo)
- Nephron Progenitor Cells
- Kidney Cortex (dissociated cells)
- Glomeruli (dissociated cells)
- Tested nuclei isolation protocol on:
- 250k Nephron Progenitor Cells - used full protocol with 2x centrifugations (500g, 5min) -> very low number of nuclei that looked good but too low to use
- 600k Glomeruli (dissociated cells) - Full protocol -> mostly debris that looked like cell clumps and that were DAPI+
- 600k Glomeruli (dissociated cells) - Just up to dounce -> same as 2, mostly debris that looked like cell clumps and that were DAPI+
- Did not see any cell pellet for the Glomeruli, maybe something happened during freezing?
- For Nephron progenitor cells, maybe the centrifugation was too low (need to use 900g 10min?)
- Need to run through my protocol with control cells to make sure it is working...