Nuclei Isolation from Frozen Tissue or Frozen hPCLS
Melanie Königshoff, Melanie Königshoff, Nayra Cardenes, Robert Lafyatis, Heidi Monroe
Abstract
This protocol follows the "Chromium Nuclei Isolation Kit" guidelines for the process for isolating Nuclei from frozen tissues and/or PCLS (Precision-Cut Lung Slices) for use in compatible 10x Genomics Single Cell assays.
Before start
- Pre-chill centrifuge to
4°C - Thaw Reducing Agent B – Thaw to
Room temperature. - Vortex – Vortex, verify no precipitate, and centrifuge briefly all Lysis and Debris Removal reagents,
- RNase Inhibitor – Centrifuge briefly.
- Buffer Preparation: Lysis Buffer & Debris Removal Buffer - Prepare the following Lysis and Debris Removal Buffers
On iceshortly before starting the Nuclei Isolation protocol. Prepare large volumes in a 15-ml or 50-ml conical tube. Vortex briefly before use. - Buffer Preparation: Wash and Resuspension Buffer - Prepare the following Wash and Resuspension Buffer
On iceshortly before starting the Nuclei Isolation protocol. Prepare large volumes in a 15-ml or 50-ml conical tube. Vortex briefly before use. - Place reagents and tubes on ice – Label tops and sides of tubes, as well as tops of spin columns, before placing
On iceand starting protocol. - Place Tissue and sample dissociation tubes on dry ice – Pre-chill on dry ice.
Attachments
Steps
Nuclei Isolation
Prepare all buffers in advance.
Transfer frozen tissue (∼50mg; use 2 slices if isolating from PCLS) to pre-chilled Sample Dissociation Tube (2000564) and place on wet ice.
Add lysis buffer (200µL) & dissociate with pestle until homogeneous while On ice.
Add lysis buffer (300µL) and pipette mix 10×. If not homogeneous, continue to dissociate with the pestle until able to pipette mix.
Incubate On ice for 0h 10m 0s.
Pipette dissociated tissue onto assembled and pre-chilled Nuclei Isolation Column and Collection Tube (2000562 & 2000563).
Centrifuge at 16000rcf,4°C .
Discard column.
Vortex flowthrough in Collection Tube for 3200rpm minimum to resuspend nuclei.
Centrifuge at 500rcf,4°C .
Remove supernatant (s/n).
Resuspend pellet with debris removal buffer (500µL).
Centrifuge at 700rcf,4°C .
Remove supernatant (s/n).
Resuspend nuclei in 1mL wash and resuspension Buffer.
Centrifuge at 500rcf,4°C .
Remove supernatant (s/n).
Repeat 15-17
Resuspend nuclei pellet in 50µL–500µL wash and resuspension Buffer.
Vortex nuclei for 0h 0m 3s and determine final nuclei concentration using AOPI or Ethidium Homodimer-1 fluorescent staining dyes and dilute if necessary for target nuclei load. Adjust nuclei concentration as necessary for intended downstream assay.
Vortex nuclei for 0h 0m 3s and keep samples On ice.
