Blue:RNA-Seq Experiments:11212016: Difference between revisions
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>B1lake (Created page with "==Kidney Samples: Nuclei isolation for Drop-seq== *Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative: #8x Dissected nephrons **Thawed and add...") |
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==Kidney Samples: Nuclei isolation for Drop-seq== | ==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 | :**Thawed and added 1ml ice cold PBS | ||
**Centrifuged at 300g for 5min | :**Centrifuged at 300g for 5min | ||
**removed most of the media and added 1ml of NEB | :**removed most of the media and added 1ml of NEB | ||
**Incubated on ice 10min and continued with dounce (5x loose pestle, 10x tight pestle) | :**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 | :**Very low count - repeated dounce tight pestle 10x - still very low count | ||
**Centrifuged and FACS - only 7200 nuclei total sorted | :**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 | :**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 | ||
'''Samples from Sanjay Jain's lab (Washington University):''' | |||
**Removed from -80C to ice, chopped using scalpel on ice (petri dish) | :*''Adult kidney tissue K1600543_1 (fresh frozen)'' | ||
**Transfered to NEB in dounce on ice | :**Removed from -80C to ice, quickly coarsely chopped using scalpel on ice (petri dish) | ||
**5 strokes with loose pestle -> ice 10 min | :**Transfered to NEB in dounce on ice | ||
**15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue | :**5 strokes with loose pestle -> ice 10 min | ||
**Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue | :**5 strokes with loose pestle | ||
**Centrifuged and combined all nuclei - re-filtered (30um filter) before FACS -> 2x 200K for DROP-seq | :**15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue -> counted nuclei as ~3M | ||
**Froze down remaining unsorted nuclei as potential bulk control | :**Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue -> counted nuclei as ~650K | ||
:**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 |
Latest revision as of 23:00, 21 November 2016
Kidney Samples: Nuclei isolation for Drop-seq[edit]
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 -> counted nuclei as ~3M
- Added another 1ml NEB to undissociated tissue -> 15 strokes with tight pestle -> removed super (Filtered 50um filter) and left undissociated tissue -> counted nuclei as ~650K
- 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)