TITLE: Ultra-deep ATAC-seq for sorted neurons
AUTHORS: Andrea Kriz, Alisa Mo

[DESCRIPTION]
Isolation of neurons from frozen post-mortem human brain tissue and preparation of ATAC-seq libraries for ultra-deep sequencing and somatic mutation detection.

[STEPS]
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
2. Buffer preparation: On the day prior to sorting, prepare the following buffers:
a. Nuclei Isolation Media (NIM): 

 

Filter through 0.22µM filter and  store at 4°C. Can keep for ~1-2 weeks. 

b. Blocking buffer: 

 

Store at 4°C. Can keep for ~1-2 weeks. 
	
c. 1X Tagmentation buffer w/ BSA for ATAC-seq:


 

Place at 4°C until ready to use. 


SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
7. Tissue preparation: 
Chill tweezers, scalpel, and dissecting plate (cell culture plate) on dry ice. Chill dounce homogenizers with B pestle on ice. Make sure swinging-bucket centrifuge is cooled to 4°C. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
11. Dounce 15-20 strokes with B pestle. If the tissue is large, use more homogenization buffer (we have used up to 5 mL) and dounce with A pestle followed by B pestle. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
12. Filter through 40µM filter into Eppendorf tube.
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
13. Spin in 4°C swinging-bucket centrifuge for 10min @ 900xg
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
14. While spin is going on, clean homogenizers by rinsing thoroughly with ddH2O, and then soaking them in 20% bleach for at least 20 minutes. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
15. Make immunostaining buffer (Blocking buffer + 1:1000 dilution NeuN-488). 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
8. Spray down all surfaces and pipettes with 70% Ethanol and DNA Away
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
9. Make homogenization buffer (NIM + additives): Homogenization buffer: 5mL NIM + 5 uL 1M DTT (1 mM final concentration) + Mini Protease inhibitor (1/2 tablet) + 25 uL 100 mM spermidine (0.5 mM final concentration). 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
10. Scrape ~10mg frozen brain tissue onto cell culture plate, either on top of dry ice or in a cryostat at -20°C. Add 1.5 mL Homogenization buffer to tissue. Add tissue/buffer mix to Dounce homogenizer. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
16. Remove supernatant. Do not disturb the pellet. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
17. Add 1mL Blocking buffer + NeuN. Resuspend pellet gently with pipette. Rotate end-to-end in the cold room for 15-20 minutes. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
18. Spin in 4°C swinging-bucket centrifuge for 5min @ 400xg
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
19. Make DAPI solution. First, dilute stock DAPI (1mg/mL) 1:15 in blocking buffer. Then, add 1 µL of the diluted dapi solution to 1 mL blocking buffer. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
20. Remove supernatant from pellet. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
21. Add 1mL Blocking buffer + Dapi and again resuspend. Strain through 40 µM filter into new Eppendorf tube immediately before nuclei sorting. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
22. Prepare tubes for ATAC-seq: Add 60 µL of 1X Tagmentation buffer into each tube for ATAC-seq (2 tubes per brain)
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
23. Sort 10,000 NeuN+ nuclei into 60µL of tagmentation buffer per ATAC-seq (2 replicates per brain sample). Proceed immediately after sorting to ATAC-seq 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
24. After sorting, spin each tube in 4 deg swinging bucket centrifuge at 500xg for 5 minutes. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
25. Remove ~75 µL of supernatant leaving  ~10 µL at bottom. Tap tube gently to mix pellet. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
26. Add 45 µL of 1X Tagmentation buffer. Do not mix.
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
27. Add 1 µL of loaded Tn5. Pipette gently 3-4 times and tap to mix. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
28. Incubate at 37°C for 30 minutes in the Thermomixer without mixing. Gently tap tube at ~10 minutes and at ~20 minutes into the incubation to mix. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
29. Stop tagmentation reaction by adding 250 µL of DNA binding buffer from Zymo Clean and Concentrator 5 kit. Add to sample tube, pipette 6x or vortex until homogenous. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
30. Follow Zymo kit instructions to purify DNA:
Add solution to spin-column tube. Spin at 10,000xg for 30 seconds. 
Wash x 200 µL wash buffer. Spin at 10,000xg for 30 seconds. 
Repeat wash x 200 µL wash buffer. Spin at 10,000xg for 30 seconds. 
Transfer spin-column tube into Eppendorf tube. Add 20 µL of water. Let sit for 1 minute. Then spin at 10,000xg for 1 minute. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
31. Prepare PCR reaction on ice:
2.5 µL i5 primer
2.5 µL i7 primer
25 µL NEBNext High-Fidelity 2X PCR Master Mix, thaw on ice and invert until homogenous
20 µL purified tagmented DNA
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
32. PCR amplification:
72°C x 5 minutes
98°C x 30 seconds
98°C x 10 seconds
63°C x 30 seconds
72°C x 1 min
Loop back to step [3] 8 times (9 cycles total)
72°C x 1 minute
4°C hold
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
33. Clean the library using Zymo clean and concentrator 5 kit. Add 250 µL of DNA binding buffer to PCR reaction and follow Zymo kit directions to purify. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
34. Elute in 40-50 µL 0.1X TE (or Zymo elution buffer). Let sit for 1 minute, spin for 1 minute. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
35. Run a 1:4 dilution of library on HS5000 Tapestation
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
37. Store libraries at -20°C.
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
3. Load Tn5: Mix 1 µL of annealed oligo A/oligo Rev with 1 µL of the annealed oligo B/oligo Rev as per Diagenode instructions. 
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
4. Add 2 µL of Diagenode Tagmentase
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
5. Incubate at RT for 30 min
SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
6. Add 2 µL glycerol. Place at -20°C until ready for use. Can store for a few week. 
SECTION: ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017))
36. 
 

SECTION: Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291)
1. Reagent list: