JAX - Nuclei Isolation for 10x Genomics A
William F Flynn, Greggory A Perry, Sandra L Daigle, Paul Gabriel, Diane Luo, Jessica Grassmann, Elise Courtois
Abstract
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the 10x Genomics Multiome assay.
This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.
This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).
Before start
Familiarize yourself with 10x Genomics protocol CG000365.
Steps
Sample Prep
Slightly thaw sample out on ice and place onto pre chilled Petri dish on ice.
Cut tissue into small pieces and add to a microcentrifuge tube.
Cell lysis
In micro centrifuge tube containing the tissue add 100µL of lysis buffer.
Using a plastic pestle to grind tissue by pushing pestle down and twisting to break up the tissue.
Start timer once lysis buffer is added.
Add 25µL to wash off pestle and place tube On ice for a total of 0h 5m 0s.
After 5 minutes of lysis On ice, 500x g,4°C.
Remove supernatant and resuspend pellet using wide bore tips in 100µL.
Incubate 4On ice for a total of 0h 5m 0s.
Washing
Add 500µL and 500x g,4°C.
Count
Count using trypan blue or AOPI to get approximate nuclei size and number.
After last wash, filter suspension through 40um filter.
Resuspend sample in 1x Nuclei buffer (10x provided) containing RNase inhibitor, and DTT.
Load
Load for transposition and continue with the 10x multiome protocol.
