Blue:RNA-Seq Experiments:11222016
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Kidney Samples: Nuclei isolation for Drop-seq[edit]
Sample from Sanjay Jain's lab (Washington University):
- Adult kidney tissue K1600543_2 (RNAlater 6hours)
- Removed from -80C to ice, thawed breifly, coarsely chopped using scalpel on ice (petri dish)
- Transfered to NEB in dounce on ice
- 5 strokes with loose pestle -> ice 10 min
- 5 strokes with loose pestle
- 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue
- Added another 1ml NEB to undissociated tissue -> ice 10min
- 15 strokes with tight pestle (tissue was tough and required extra force and twisting at bottom to disrupt) -> removed super (Filtered 50um filter)
- Centrifuged and combined all nuclei - re-filtered (30um filter) before FACS -> sort 2 aliquots of 200K for DROP-seq
- Froze down remaining unsorted nuclei as potential bulk control
- Adult kidney tissue K1600543_2 (RNAlater 6hours)
Note: RNAlater seems to make the tissue tougher... may need to chop more finely before adding to NEB next time. Hopefully there is no bias in the tissue type that is getting effectively dissociated to release nuclei