TITLE: Nuclei isolation from snap-frozen human placental tissue for bulk ATACseq
AUTHORS: Scott   Lindsay-Hewett

[DESCRIPTION]
This protocol describes the isolation of nuclei from snap-frozen human placental tissue for bulk ATACseq. It is a modified version of a protocol (SCBL Protocols - 10x Multiome Nuclei Isolation) supplied by the Jackson Laboratory (JAX), which is itself modified from Sigma Aldrich's Nuclei Isolation Kit: Nuclei EZ Prep protocol, and 10x Genomics' Demonstrated Protocol – Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing. It is recommended to work with no more than four samples at a time so that all samples can be processed in a timely manner.

[STEPS]
SECTION: Preparation
1. Pre-chill microfuge to 4 °C.
SECTION: Preparation
2. Prepare bucket of ice, and chill metal plate (allows for a flat surface to place Petri dish on top of - to keep tissue cool while cutting).
SECTION: Preparation
4. Thaw 110 µL aliquot of 100 millimolar (mM) and place on ice.
 

SECTION: Make fresh buffers
9. Prepare 1 mL as detailed below, and keep on ice:

988 µL 
2 µL 
10 µL 
 


SECTION: Make fresh buffers
10. Prepare 8.5 mL as detailed below, and keep on ice:

85 µL 
17 µL 
25.5 µL 
850 µL 
85 µL 
85 µL 
7352.5 µL 
 


SECTION: Preparation
5. Thaw 500 µL aliquot of tagmentation buffer and place on ice.
 

 

SECTION: Preparation
3. Dispense 200 µL into microfuge tube and place on ice

SECTION: Preparation
8. Get the following materials ready; you won't have time to do this in between steps:

4 x #22 disposable scalpels
4 x 60mm Petri dishes
4 x disposable pestles/tubes, along with cordless motor
4 x disposable tweezers
4 x 40um filters
4 x 20um filters
4 x BioRad counting slides
0.4% Trypan Blue stain
200ul wide bore filter tips
1000ul wide bore filter tips
SECTION: Nuclei isolation
11. Place a sterile Petri dish onto the chilled metal plate sitting on ice. Move tube containing first placenta sample from dry ice to wet ice for ~30 seconds, then tap slightly-thawed tissue into pre-chilled Petri dish.
SECTION: Preparation
6. Pull snap-frozen placenta samples (~150-200 mg each) from -80 °C  and place on dry ice until ready to process.
 
  

SECTION: Nuclei isolation
12. Cut tissue into small pieces (~ 2 to 3mm in size) using a fresh disposable #22 scalpel. Use disposable tweezers to hold tissue in place while cutting. Transfer tissue into labeled pre-chilled microfuge tube.
 

SECTION: Preparation
7. Label 4 microfuge tubes (the ones that are supplied with the disposable pestles) with sample names and pre-chill on ice.
SECTION: Nuclei isolation
13. Add 125 µL and grind tissue with a disposable pestle attached to a cordless motor.
 


SECTION: Nuclei isolation
14. Place ground-up tissue on ice for 5 min. During this time you can start step 11 for the second placenta sample.

SECTION: Nuclei isolation
15. Centrifuge sample 500 x g, 5 min, 4 °C. During this time you can start step 11 for the third placenta sample.

SECTION: Nuclei isolation
16. Remove and discard supernatant, and resuspend pellet gently in 100 µL using 200ul wide bore filter tips. Incubate on ice for 5 min. During this time you can start step 11 for the fourth placenta sample.

SECTION: Nuclei isolation
17. Using a 1000ul wide bore filter tip, add 500 µL, mix gently, and centrifuge 500 x g, 5 min, 4 °C.
SECTION: Nuclei isolation
18. Remove and discard supernatant and repeat wash twice, saving the last suspension on ice until all samples reach this step.
SECTION: Nuclei isolation
19. Filter each sample though a 40um filter into a fresh microfuge tube. To maximize recovery of nuclei, wash old microfuge tube with 200 µL, and pass through the same 40um filter.

SECTION: Nuclei isolation
20. Repeat previous step, this time with a 20um filter. This filter will remove debris that wasn't removed in the previous step.
SECTION: Nuclei isolation
21. Centrifuge 500 x g, 5 min, 4 °C.

SECTION: Nuclei isolation
22. Remove and discard supernatant carefully, and gently resuspend pellet in 50 µL using 200ul wide bore filter tip. Clumps of nuclei should break up easily. Place resuspended nuclei on ice.

SECTION: Nuclei isolation
23. Take 5 µL aliquot of resuspended nuclei; add 20 µL and 25 µL. Flick to mix, then load 10 µL to both sides of a BioRad counting slide. Determine nuclei concentration using an automated cell counter.
 


SECTION: Nuclei isolation
24. Based on nuclei concentration, prepare 10 µL of a 5,000 nuclei/ul solution (50,000 total nuclei), using tagmentation buffer for dilution. Proceed immediately to tagmentation and library preparation.
 


SECTION: Post-steps
25. Centrifuge remaining nuclei from step 22 500 x g, 5 min, 4 °C. Remove and discard supernatant, and gently resuspend nuclei in 50 µL using 200ul wide bore filter tips, making sure to break up clumps of nuclei. Store nuclei at -80 °C for up to several months.