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:
  1. Isolated Nephrons (intact X8; see attached photo)
  2. Nephron Progenitor Cells
  3. Kidney Cortex (dissociated cells)
  4. Glomeruli (dissociated cells)
  • Tested nuclei isolation protocol on:
  1. 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
  2. 600k Glomeruli (dissociated cells) - Full protocol -> mostly debris that looked like cell clumps and that were DAPI+
  3. 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...