Blue:RNA-Seq Experiments:12052016: Difference between revisions
Jump to navigation
Jump to search
>B1lake |
>B1lake |
||
Line 29: | Line 29: | ||
:**15 strokes with tight pestle | :**15 strokes with tight pestle | ||
:**Centrifuged combined nuclei - re-filtered (30um filter) before FACS -> 0.6M nuclei | :**Centrifuged combined nuclei - re-filtered (30um filter) before FACS -> 0.6M nuclei | ||
:**Sorted 200K for DROP-seq | :**Sorted 200K for DROP-seq - post-centrifuge ~125K | ||
:**Froze down remaining unsorted nuclei as potential bulk control | :**Froze down remaining unsorted nuclei as potential bulk control |
Latest revision as of 23:05, 5 December 2016
PDX Single-Cell RNA Seq[edit]
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
- Note: really bad clumping after centrifuging - count was only ~32K. Therefore, only enough to run Drop-seq and too clumping to run on C1
- Froze down remaining unsorted nuclei as potential bulk control
Note: Tissue was very amendable to dissociation
Kidney Samples: Nuclei isolation for Drop-seq[edit]
Samples from Sanjay Jain's lab (Washington University):
- Adult kidney tissue sections (40um) K1600543FBSLC
- Isolated nuclei:
- 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 - post-centrifuge ~125K
- Froze down remaining unsorted nuclei as potential bulk control