TITLE: Simplified ATAC-seq protocol on frozen brains of fruit flies
AUTHORS: Ryan Corces, William J. Greenleaf, Howard Y. Chang, Siyuan Feng, Jamie Freeman

[DESCRIPTION]
This protocol enables the isolation of nuclei from frozen tissues. These nuclei are suitable for use in ATAC-seq, single-cell ATAC-seq, ChIP-seq, HiC/3C, and many other assays.

Modified for a small amount of fruit fly brains from the omni-ATACseq protocol from Ryan Cortes

[GUIDELINES]
The quality of the tissue at the time of freezing is a major factor in the quality of data downstream. If freezing tissue for use later, you may want to consider cryopreserving 50 mg tissue chunks in BAM Banker cryopreservative. If using previously frozen tissue, the ischemic time or post mortem interval should be kept as short as possible.

Nuclei can be cryopreserved in BAM Banker. In our experience, freeze-thaw cycles do not adversely affect data quality as long as thawing is carried out on ice and freezing is performed using a slow-freeze freezing container. We have had success isolating nuclei from every tissue we have tried including difficult to work with tissues such as heart.

[STEPS]
SECTION: Before you start the protocol:
1. All steps should be performed on ice or at 4 °C. Pre-chill a fixed angle centrifuge to 4°C.

SECTION: Before you start the protocol:
2. Pre-chill all tubes. For each sample you are processing, you will need: (i) one 2 ml cryo tube with 10 1.6 mm beads for bead homogenization (ii)  one 1.5 ml round-bottom LoBind tube for nuclei pelleting (iii) one 1.5 ml round-bottom LoBind tube for collecting purified DNA 
SECTION: Before you start the protocol:
3. Prepare all buffers. For faster dissolution, crush protease inhibitor tablets prior to addition to 1x Homogenization Buffer Unstable Solution. DTT, Spermidine, Spermine, and digitonin are stored at -20°C. All other detergents, ATAC-RSB, and other buffers are stored at 4 °C. Do not prepare transposition mix ahead of time.

SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
4. move samples from liquid nitrogen, or -80 °C  freezer  onto on ice wet ice 2 mins before homogenization. Add 200 µL  HB to the tube wall, vortex and spin down to get the sample off the tube wall. 

SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
5. Homogenize in BeadRupter Elite bead mill homogenizer using 2 cycles of the following:
	Speed 1.0 for 20 s
	Dwell for 20 s
	Speed 2.0 for 20 s
	Dwell for 20 s
SECTION: DNA Library Amplification
20. Place tubes in thermal cycler, making sure lid is tightly against your tubes, and run program ML2.
SECTION: DNA Library Amplification
19. Add 10 uL of cleaned tagmentation product. Mix and spin down. Make sure to close PCR tube lids tightly with a marker end.
SECTION: DNA Library Amplification
18. Add 2 uL of primer mix to each reaction. Make sure to record which index primers are used!
SECTION: DNA Library Amplification
17. Visually inspect primer plate to ensure evaporation has not occurred in the wells you intend to use. Spin down in plate spinner. Carefully open foil to needed wells.
SECTION: DNA Library Amplification
16. Mix and spin down master mix, keep cold, and add 38 uL to each reaction tube.


SECTION: DNA Library Amplification
15. Remove Hifi enzyme from freezer and place on wet ice. Make PCR master mix (MM)

PCR master mix (MM)

 

SECTION: DNA Library Amplification
14. Remove PCR reagents from freezer to thaw at Room temperature; dNTPs, 2X KAPA High fidelity buffer (yellow lid), and primer plate.  Once all traces of ice are gone, vortex and spin down. Turn on thermal cycler.

SECTION: (Optional) Cleanup and Freezing Down of Extra Nuclei
21. If you would like to save extra nuclei for other assays or to potentially use in additional ATAC-seq experiments downstream:
SECTION: Transposition of Nuclei
12. After incubation, follow instructions of Qiage Minelute DNA clean-up Kit to purify the DNA
SECTION: Transposition of Nuclei
11. Incubate reactions at 37 °C for 30 min in a heat block (ideally should use a thermoshaker with 1000 RPM constant shaking).


SECTION: Transposition of Nuclei
10.1. Unlike the published ATAC-seq protocols, you do not need to do an individual lysis step in this protocol because the nuclei are exposed to NP40 throughout the Douncing portion of the protocol.
SECTION: Transposition of Nuclei
10. Add 50 ul ATAC-seq Reaction Mix to each tube and pipette up and down 6 times to resuspend nuclei pellet.
SECTION: Transposition of Nuclei
9. Using a p1000 pipette, remove all but the last 100 ul of supernatant. Remove last 100 ul with p200 pipette set to 200 ul using a single fluid pipetting motion. Place the tip of your pipette on the opposite side of the tube to where the nuclei pellet is located during this final aspiration step.
8. Centrifuge nuclei for 10 min at 500 RCF at 4 °C in a fixed angle centrifuge. At this point, the pellet should be clearly visible if 50,000 nuclei were used. Pellets of as few as 10,000 nuclei should be visible.


SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
6. Pellet nuclei by spinning 5 min at 4 °C at 350 RCF in a fixed angle centrifuge.


SECTION: Transposition of Nuclei
13. Store purified DNA under-20 °C , if not used for PCR for a more than a week. Otherwise the purified DNA can be stored at 4 °C  temporarily

SECTION: DNA Library Amplification
17.1. primer plate in use:

 


SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
4.1. if samples were slow-frozen in Bambanker, put the sample on on ice until the Bambanker thaws, then spin down sample  5 min at 4 °C at 750 RCF in a fixed angle centrifuge, and remove supernatant. Add 200 µL  HB and 10 1.6 mm beads to the tube. 
SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
4.2. if samples are fresh, put the sample on  on on ice until homogenization (200 µL  pre-chilled HB should have been added to the tube before dissection. 
SECTION: Isolation of Nuclei via Bead Homogenization and Centrifuging
7. Re-suspend the nuclei in 1000 µL of ATAC-RSB-Tween Buffer