TITLE: Nuclei Isolation from Frozen Tissue or Frozen hPCLS
AUTHORS: Heidi Monroe, Nayra Cardenes, Melanie Königshoff, koenigshoffm, Robert Lafyatis

[DESCRIPTION]
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 ice shortly 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 ice shortly 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 ice and starting protocol.
Place Tissue and sample dissociation tubes on dry ice – Pre-chill on dry ice.


[STEPS]
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3. Add lysis buffer (200 µL) & dissociate with pestle until homogeneous while on ice.

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2. 
 

Transfer frozen tissue (∼50 mg; use 2 slices if isolating from PCLS) to pre-chilled Sample Dissociation Tube (2000564) and place on wet ice.

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4. 
 

Add lysis buffer (300 µL) and pipette mix 10×. If not homogeneous, continue to dissociate with the pestle until able to pipette mix.

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5. Incubate on ice for  10 min.

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6. Pipette dissociated tissue onto assembled and pre-chilled Nuclei Isolation Column and Collection Tube (2000562 & 2000563).

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7. Centrifuge at 16000 rcf, 20 s, 4 °C .

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8. Discard column.
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9. Vortex flowthrough in Collection Tube for 3200 rpm, 10 s minimum to resuspend nuclei.

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16. Centrifuge at 500 rcf, 5 min, 4 °C .

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15. Resuspend nuclei in 1 mL wash and resuspension Buffer.

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14. Remove supernatant (s/n).

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13. Centrifuge at 700 rcf, 10 min, 4 °C .

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12. Resuspend pellet with debris removal buffer (500 µL).

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11. Remove supernatant (s/n).

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10. Centrifuge at 500 rcf, 3 min, 4 °C .

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19. Resuspend nuclei pellet in 50 µL–500 µL wash and resuspension Buffer.

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17. Remove supernatant (s/n).

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18. Repeat 15-17

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20. Vortex nuclei for 3 s 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. 

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21. Vortex nuclei for 3 s and keep samples on ice.
 


SECTION: Nuclei Isolation
1. Prepare all buffers in advance.