Blue:RNA-Seq Experiments:04272017

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Fetal Kidney Samples: Nuclei isolation for Drop-seq[edit]

Samples from McMahon's lab (USC):

  • 13 week whole glomeruli in RNAlater
  • 13 week biopsy kidney punch in RNAlater
  • 15 week whole glomeruli in RNAlater
  • 15 week biopsy kidney punch in RNAlater
  • Isolated nuclei:
    • Removed from -80C to RT to thaw, centrifuged at 3000 g (13 week) or 4500 g (15 week), removed super and added NEB -> ice 10 min
    • 5 strokes with loose pestle
    • 15 strokes with tight pestle
    • Removed super (Filtered 50um filter) and added 9ml PBSE
    • Centrifuged - re-filtered (30um filter) before FACS
  • Note:
    • 13 week glomeruli had low yield (50K) while biopsy had good yield (500K), combined for FACS -> 180K final post-sort
    • 15 week glomeruli had good yield (120K) while biopsy had no yield (0K), combined just in case for FACS -> 30K final post-sort
    • maybe 4500 g for tissue is too much...