Nuclear extraction from endometrial tumors for single nuclei sequencing
gracefoley
Abstract
Modified from: Slyper, M. et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat. Med. 2020 265 26 , 792–802 (2020).
A optimized protocol for nuclear extraction from endometrial tumors. Performed with endometrial adenocarcinoma, endometrioid type, FIGO grade 1.
Nuclear extraction optimization for single nuclei RNA-seq.pptx
Steps
Place frozen tissue in a well of a 6 well plate containing 1 mL of cold NST disassociation buffer.
Ensure plate is on ice
Chop tissue using a scalpel until mostly homogenized
Incubate solution on ice for 5 minutes
Filter the homogenized solution through a 40 uM falcon cell strainer into a 50 mL falcon tube
Use an additional 1 mL of NST buffer to rinse the well and filter
Count nuclei
Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
Resuspend pellet and bring the volume up to 5 mL using ST buffer
Transfer solution to 15 mL conical tube and centrifuge for 10 minutes at 300 g at 4oC
- Resuspend pellets on ice in 1 ml of the ST buffer
- Filter through 35 um falcon strainer
- Count nuclei
Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
- Resuspend nuclei pellet in resuspension buffer to get a concentration of 1,000 nuclei per uL
Start with .5 mL per 0.2 grams of endometrial tumor tissue
Look at nuclear membranes at 40x-60x to ensure high nuclear membrane quality with minimal blebbing
