Blue:RNA-Seq Experiments:11212016: Difference between revisions

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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:
'''Samples from Andy Ransick (McMahon lab, USC) gently frozen in 1ml cryopreservative:'''
#8x Dissected nephrons
:*''8x Dissected nephrons''
**Thawed and added 1ml isce cold PBS
:**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):
 
#Adult kidney tissue K1600543_1 (fresh frozen)
'''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 (~3M nuclei)
:**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 (~650K nuclei)
:**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


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