Blue:RNA-Seq Experiments:12052016: Difference between revisions
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>B1lake (Created page with "===PDX Single-Cell RNA Seq=== '''Meduloblastoma tissue (PDX) from Weschsler-Reya Lab''' *Tissue was flash frozen on 10/19/2016 (3 separate chunks) *Isolated nuclei: :**Remove...") |
>B1lake No edit summary |
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''Note: Tissue was very amendable to dissociation'' | ''Note: Tissue was very amendable to dissociation'' | ||
==Kidney Samples: Nuclei isolation for Drop-seq== | |||
'''Samples from Sanjay Jain's lab (Washington University):''' | |||
:*''Adult kidney tissue sections (40um) K1600543FBSLC'' | |||
:**Removed from -80C to ice, quickly added NEB and transferred to dounce | |||
:**5 strokes with loose pestle -> ice 10 min | |||
:**15 strokes with tight pestle -> ice 10 min | |||
:**Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue | |||
:**15 strokes with tight pestle -> ice 10 min | |||
:**Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue | |||
:**15 strokes with tight pestle | |||
:**Centrifuged combined nuclei - re-filtered (30um filter) before FACS -> 0.6M nuclei | |||
:**Sorted 200K for DROP-seq | |||
:**Froze down remaining unsorted nuclei as potential bulk control |
Revision as of 20:33, 5 December 2016
PDX Single-Cell RNA Seq
Meduloblastoma tissue (PDX) from Weschsler-Reya Lab
- Tissue was flash frozen on 10/19/2016 (3 separate chunks)
- Isolated nuclei:
- Removed from -80C, transferred quickly to NEB and used scalpel to mash, transferred to dounce
- 5 strokes with loose pestle
- 15 strokes with tight pestle -> ice 10 min
- Filtered 50um filter
- Centrifuged - re-filtered (30um filter) before FACS -> 5M nuclei
- Sorted 200K for DROP-seq
- Froze down remaining unsorted nuclei as potential bulk control
Note: Tissue was very amendable to dissociation
Kidney Samples: Nuclei isolation for Drop-seq
Samples from Sanjay Jain's lab (Washington University):
- Adult kidney tissue sections (40um) K1600543FBSLC
- Removed from -80C to ice, quickly added NEB and transferred to dounce
- 5 strokes with loose pestle -> ice 10 min
- 15 strokes with tight pestle -> ice 10 min
- Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue
- 15 strokes with tight pestle -> ice 10 min
- Removed super (Filtered 50um filter) and added 1ml NEB to undissociated tissue
- 15 strokes with tight pestle
- Centrifuged combined nuclei - re-filtered (30um filter) before FACS -> 0.6M nuclei
- Sorted 200K for DROP-seq
- Froze down remaining unsorted nuclei as potential bulk control
- Adult kidney tissue sections (40um) K1600543FBSLC