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...