Blue:RNA-Seq Experiments:11212016: Difference between revisions
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:**Transfered to NEB in dounce on ice | :**Transfered to NEB in dounce on ice | ||
:**5 strokes with loose pestle -> ice 10 min | :**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 (~3M nuclei) | :**15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue (~3M nuclei) | ||
:**Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue (~650K nuclei) | :**Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue (~650K nuclei) | ||
:**Centrifuged and combined all nuclei - re-filtered (30um filter) before FACS -> 2x 200K for DROP-seq | :**Centrifuged and combined all nuclei - re-filtered (30um filter) before FACS -> 2x 200K for DROP-seq | ||
:**Froze down remaining unsorted nuclei as potential bulk control | :**Froze down remaining unsorted nuclei as potential bulk control |
Revision as of 22:59, 21 November 2016
Kidney Samples: Nuclei isolation for Drop-seq
Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative:
- 8x Dissected nephrons
- Thawed and added 1ml ice cold PBS
- Centrifuged at 300g for 5min
- removed most of the media and added 1ml of NEB
- Incubated on ice 10min and continued with dounce (5x loose pestle, 10x tight pestle)
- Very low count - repeated dounce tight pestle 10x - still very low count
- Centrifuged and FACS - only 7200 nuclei total sorted
- Centrifuged and resuspended in 10 ul - only 80/ul final -> too low to run on Drop-seq or C1 -> froze down as potential bulk control
- 8x Dissected nephrons
Samples from Sanjay Jain's lab (Washington University):
- Adult kidney tissue K1600543_1 (fresh frozen)
- Removed from -80C to ice, quickly 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 (~3M nuclei)
- Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue (~650K nuclei)
- Centrifuged and combined all nuclei - re-filtered (30um filter) before FACS -> 2x 200K for DROP-seq
- Froze down remaining unsorted nuclei as potential bulk control
- Adult kidney tissue K1600543_1 (fresh frozen)