Editing
Blue:RNA-Seq Experiments:11212016
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
==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 '''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
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information