Jul 09, 2018
Human Kidney / Tumour Tissue Disaggregation for Single Cell RNA Sequencing (10x Genomics platform)
- Kevin Loudon1,
- John Ferdinand1,
- Alexandra Riding1,
- Menna Clatworthy1
- 1University of Cambridge
Protocol Citation: Kevin Loudon, John Ferdinand, Alexandra Riding, Menna Clatworthy 2018. Human Kidney / Tumour Tissue Disaggregation for Single Cell RNA Sequencing (10x Genomics platform). protocols.io https://dx.doi.org/10.17504/protocols.io.qtrdwm6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 08, 2018
Last Modified: July 09, 2018
Protocol Integer ID: 12881
Materials
STEP MATERIALS
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Protocol materials
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Tissue Preparation
Tissue Preparation
Take dissected tissue (renal cortex, medulla or tumour) and weigh tissue (typical biopsy size used 0.5 - 1 gram)
Pour approximately 2-3 mLs of "Digest Mix" onto sampe in 10cm3 petridish
Note
PREPARATION OF DIGEST MIX
Ingredients
(1) RPMI alone
(2) Liberase TM (Sigma Aldrich)
(3) DNAse (Sigma Aldrich)
For 50mLs of RPMI add:
--> 625 microlitres of Liberase (Stock solution 2.5mg/mL)
--> 250 microlitres of DNAse (Stock solution 0.05mg/mL)
Using a razor blade mince into small pieces approximately 2mm3 .
Transfer tissue into a gentleMACS C tube and add further 3-4 mLs of Digest mix.
Note
GentleMACS C tube by Miltenyi Biotec (Cat.130-096-334)
Place in shaking incubator at 37oC for 30 minutes.
Homogenise sample in GentleMACS tube using program "Spleen 4" and "Lung 2" on GentleMACS dissociator.
Pass through a 100µm cell strainer with of a 2.5ml syringe plunger and wash through with cold running buffer.
Note
PREPARATION OF RUNNING BUFFER
Ingredients (for 1 litre)
(1) 1L PBS
(2) 5ml BSA (from reagent diluent kit)
(2) 4ml 0.5M EDTA
Centrifuge in a bench top centrifuge at 2000 RPM for 10 minutes and CAREFULLY remove the supernatant.
If sample is contaminated with red blood cells an additional red cell lysis step can be taken.
To ensure optimal yield for 10X Genomics single cell platform, a live cell enrichment step is required - this was performed using Miltenyi 'Dead Cell Removal Kit' (Please see manufacturers instructions for further details).
Note
LIVE CELL ENRICHMENT (Miltenyi - Dead Cell Removal Kit)
Ingredients
(1) Dead Cell removal Kit - Miltenyi (Order No. 130-090-101)
(2) MACS Column (LS or MS)
In brief for MACS colum LS
(1) Use LS column for 10^8 dead cells or 10^9 total cells.
(2) Remove supernatant completely following previous steps
(3) Resuspend pellet in 100 µL of 'Dead Cell Removal MicroBeads' per approximately 10^7 total cells.
(4) Incubate 15 minutes at room temperature (20–25 °C).
(5) Rinse column with 1x binding buffer as per manufacturers instructions.
(6) Apply cell suspension in 1-10mLs of binding buffer and collect the effluent as the NEGATIVE cell population (i.e the live cells).
(7) Wash cells with PBS for 5 minutes at 1500rpm.
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
Count the cells and resuspend the live cell supsension in appropriate volume of PBS for the 10X application.