Jun 06, 2024
Version 1
Simplified ATAC-seq protocol on frozen brains of fruit flies V.1
This protocol is a draft, published without a DOI.
- Ryan Corces1,
- William J. Greenleaf2,
- Howard Y. Chang2,
- Siyuan Feng3,
- Jamie Freeman4
- 1Gladstone Institutes;
- 2Stanford University;
- 3University of Wisconsin-Madison;
- 4University of Wisconsin Madison
- Human Cell Atlas Method Development Community
External link: https://www.ncbi.nlm.nih.gov/pubmed/28846090
Protocol Citation: Ryan Corces, William J. Greenleaf, Howard Y. Chang, Siyuan Feng, Jamie Freeman 2024. Simplified ATAC-seq protocol on frozen brains of fruit flies. protocols.io https://protocols.io/view/simplified-atac-seq-protocol-on-frozen-brains-of-f-daw72fhn
Manuscript citation:
Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, Satpathy AT, Rubin AJ, Montine KS, Wu B, Kathiria A, Cho SW, Mumbach MR, Carter AC, Kasowski M, Orloff LA, Risca VI, Kundaje A, Khavari PA, Montine TJ, Greenleaf WJ†, Chang HY†. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. 2017. Nature Methods. (PMID: 28846090).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 20, 2024
Last Modified: June 06, 2024
Protocol Integer ID: 96959
Keywords: ATAC-seq, Nuclei Isolation, Frozen Tissue Homogenization
Abstract
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.
Materials
STOCK BUFFER INGREDIENTS
| A | B | C | D | E | F | G | H | I | J | |
| Stock (planned Conc.) | Name | chemical info | Formula mass (g/mol) | Volume (ml) of solution (planned) 2024.03.20 | Mass (g) of solube (planned) 2024.03.20 | Volume (ml) of solution (actual) 2024.03.20 | Mass (g) of solube (actual) 2024.03.20 | Actual Conc. (M) 2024.03.20 | autoclave bottle before usage | |
| 1 | M Sucrose | 342.30 | 100 | 34.23 | 100 | 34.34 | 1.0032135553607946 | 1 | ||
| 2 | M KCl | BDH, potassium chloride, purity 99 - 100.5%, lot No. 118446, opened 2024.03.20 | 74.47 | 100 | 14.894 | 100 | 15.37 | 2.0639183563851216 | 1 | |
| 1 | M MgCl2 (hexahydrate) | dot scientific inc., magnesium Chloride hexahydrate, purity unknown, lot No. 61024-91729, opened 2019.06.18 | 203.3 | 100 | 20.33 | 100 | 20.44 | 1.0054107230693556 | 1 | |
| 0.75 | M Tricine-KOH pH 7.8 | 179.22 | 200 | 26.883 | 150 | 13.38 | 0.4977123088940967 | 1 | ||
| 2.5 | M NaCl | Fisher Scientific, Sodium Chloride, putiry 99.3%, log No. 145475, already opened 2024.03.20 | 58.44 | 200 | 29.22 | 250 | 29.22 | 2 | 1 | |
| 60 | % Iodixanol | |||||||||
| 0.5 | M Spermidine | 254.63 | 2 | 0.25463 | 2 | 0.257 | 0.504653811412638 | |||
| 0.15 | M Spermine | 202.34 | 8 | 0.242808 | 8 | 0.2411 | 0.14894484530987448 | |||
| 1 | M DTT | 154.25 | 3 | 0.46275 | 3 | 0.468 | 1.0113452188006482 | |||
| 10 | % wt./vol. Tween-20 | opened 2024.04.02 | density: 1.095 g/mL at 25 °C | 50 | ||||||
| 10 | % wt./vol. IGEPAL® CA-630 | opened 2024.04.04 | density: 1.06 g/mL at 25 C | 50 |
STOCK BUFFER INGREDIENTS (dilute by density)
| A | B | C | D | E | F | G | H | I | J | K | L | M | |
| Stock (planned Conc.) | Name | chemical info | Density (g/mL at 25 C) | Volume (ml) of solution (planned) 2024.04.04 | Mass (g) of solube (planned) 2024.04.04 | Volume (mL) of solube (planned) 2024.04.04 | Volume (mL) of water (planned) 2024.04.04 | Mass (g) of solube (actual) 2024.04.04 | Volume (mL) of solube (actual) 2024.04.04 | Volume (mL) of water (actual) 2024.04.04 | Sterile filter? | ||
| 10 | % wt./vol. Tween-20 | opened 2024.04.02 | 1.095 | 50 | 5 | 4.566210045662101 | 45.4337899543379 | 5.005 | 4.570776255707763 | 45.42922374429224 | yes | ||
| 10 | % wt./vol. IGEPAL® CA-630 | opened 2024.04.04 | 1.06 | 50 | 5 | 4.716981132075471 | 45.283018867924525 | 5.005 | 4.721698113207546 | 45.278301886792455 | yes |
STOCK BUFFERS
All stock solutions should be filtered using a 0.22 um PVDF filter system. All solutions except for the 50% Iodixanol solution are stable at 4°C for at least 6 months.
1.034x Homogenization Buffer Stable Solution - 200 ml (2014 ul/sample)
| A | B | C | D | E | F | |
| Stock | Name | Final Conc. | Actual stock conc.(made on 2024.03.20) | actual Fold Dilution (x) | actual Total Vol. (ml) | |
| 1 | M Sucrose | 0.26 | 1.00 | 3.846153846153846 | 52.00000000000001 | |
| 2 | M KCl | 0.03 | 2.06 | 68.66666666666667 | 2.9126213592233006 | |
| 1 | M MgCl2 | 0.01 | 1.01 | 101 | 1.9801980198019802 | |
| 0.75 | M Tricine-KOH pH 7.8 | 0.02 | 0.50 | 25 | 8 | |
| - | Water | - | - | - | 135.1071806209747 |
Diluent Buffer - 100 ml (16.4 ul/sample)
| A | B | C | D | E | F | |
| Stock | Name | Final Conc. | Actual stock conc.(made on 2024.03.20) | actual Fold Dilution (x) | actual Total Vol. (ul) | |
| 2 | M KCl | 0.15 | 2.06 | 13.733333333333334 | 7.281553398058252 | |
| 1 | M MgCl2 | 0.03 | 1.01 | 33.66666666666667 | 2.97029702970297 | |
| 0.75 | M Tricine-KOH, pH 7.8 | 0.12 | 0.50 | 4.166666666666667 | 24 | |
| - | Water | - | - | - | 65.74814957223879 |
50% Iodixanol Solution - 50 ml (Remake monthly for stability) (840 ul/sample)
| A | B | C | D | E | |
| Stock | Name | Final Conc. | Fold Dilution (x) | Total Vol. (ml) | |
| - | Diluent Buffer | 1 | - | 8.33333 | |
| 60 | % Iodixanol | 50 | 1.20 | 41.66667 |
ATAC-RSB Buffer - 500 ml (2970 ul/sample)
| A | B | C | D | E | F | |
| Stock | Name | Final Conc. | Actual stock conc.(made on 2024.03.20) | actual Fold Dilution (x) | actual Total Vol. (ml) | |
| 1 | M Tris-HCl pH 7.5 | 0.01 | 0.50 | 50 | 10 | |
| 5 | M NaCl | 0.01 | 2 | 200 | 2.5 | |
| 1 | M MgCl2 | 0.003 | 1.01 | 336.6666666666667 | 1.4851485148514851 | |
| - | Water | - | - | - | 486.01485148514854 |
SAME DAY BUFFERS
1x Homogenization Buffer Unstable Solution
**Note – cOmplete Protease Inhibitors come as tablets. It is difficult to use less than 1/2 tablet so we prepare the 1x Homogenization Buffer Unstable Solution in batches of 12 as outlined below.
| A | B | C | D | E | F | G | |
| Stock | Name | Final Conc. | Fold Dilution (x) | Vol per 12 samp. (ul) | Vol (ul) made 2024.04.24 (14.4 sample) | Vol (ul) made 2024.05.01 (10 sample, assuming 200 ul per sample) | |
| 1.0341 | x HB Stable Solution | 1 | 1.03 | 24175.00 | 29010 | 2417.5 | |
| 1 | M DTT | 0.001 | 1000.00 | 25.00 | 30 | 2.5 | |
| 500 | mM Spermidine | 0.5 | 1000.00 | 25.00 | 30 | 2.5 | |
| 150 | mM Spermine | 0.15 | 1000.00 | 25.00 | 30 | 2.5 | |
| 10 | % NP40 | 0.3 | 33.33 | 750.00 | 900 | 75 | |
| - | cOmplete Protease Inhibitor mini tablets | - | - | 2.50 Tablets | 3 tablets | 0.25 tablet | |
| TOTAL VOLUME | 25000 | 30000 | 2500 |
30% Iodixanol Solution
| A | B | C | D | E | F | |
| Stock | Name | Final Conc. | Fold Dilution (x) | Vol per sample (ul) | Vol (ul) made 2024.04.04 (12 samp.) | |
| - | 1x Homog. Buffer Unstable | - | - | 240.00 | 2880 | |
| 50 | % Iodixanol Solution | 30 | 1.67 | 360.00 | 4320 |
40% Iodixanol Solution
| A | B | C | D | E | F | |
| Stock | Name | Final Conc. | Fold Dilution (x) | Vol per sample (ul) | Vol (ul) made 2024.04.04 (12 samp.) | |
| - | 1x Homog. Buffer Unstable | - | - | 120.00 | 1440 | |
| 50 | % Iodixanol Solution | 40 | 1.25 | 480.00 | 5760 |
ATAC-RSB-Tween Buffer
| A | B | C | D | E | F | G | |
| Stock | Name | Final Conc. | Fold Dilution (x) | Vol per sample (ul) | Vol (ul) made 2024.04.04 (12 samp.) | Vol (ul) made 2024.05.01 (11 samp., assuming 1000 ul per sample) | |
| - | ATAC-RSB | - | - | 2970.00 | 35640 | 10890 | |
| 10 | % Tween-20 | 0.1 | 100.00 | 30.00 | 360 | 110 |
ATAC-seq Reaction Mix
| A | B | C | D | |
| Reagent | Vol per sample (ul) | Vol (ul) made 2024.04.24 (11 samp.) | Vol (ul) made 2024.05.01 (11 samp.) | |
| H2O | 5.25 | 57.75 | 57.75 | |
| PBS | 16.5 | 181.5 | 181.5 | |
| 2x TD Buffer | 25 | 275 | 275 | |
| 2% Digitonin | 0.25 | 2.75 | 2.75 | |
| 10% Tween-20 | 0.5 | 5.5 | 5.5 | |
| Tn5 Transposase | 2.5 | 27.5 | 27.5 |
PCR master mix (MM)
| A | B | C | D | |
| Reagent | Vol per sample 1x50 ul run (ul) | Vol (ul) made 2024.04.25 (10 + 1 samp.) | Vol (ul) made 2024.05.05 (10 + 1 + 1 samp.) | |
| H2O | 25.5 | 280.5 | 306 | |
| 2 X KAPA high fidelity butter | 10 | 110 | 120 | |
| dNTPs | 1.5 | 16.5 | 18 | |
| Hifi enzyme | 1 | 11 | 12 | |
| TOTAL MM VOLUME | 38 | 418 | 456 |
Reagents used in this protocol
| A | B | C | |
| Item | Supplier | Cat Number | |
| Eppendorf 2 ml Lo-Bind tubes | Sigma | Z666556-250EA | |
| Eppendorf 1.5 ml Lo-Bind tubes | Sigma | Z666548-250EA | |
| Nunc cryovials | Thermo | 375418PK | |
| Iodixanol (comes at 60%) | Sigma | D1556-250ML | |
| Digitonin | Promega | G9441 | |
| Sucrose | Sigma | S7903-250G | |
| IGEPAL® CA-630 (identical with NP40, which was discontinued) | Sigma | I8896-50ML | |
| Tricine | Sigma | T0377-25G | |
| Potassium Hydroxide (KOH) | Sigma | P5958-250G | |
| cOmplete Protease Inhibitors mini tablets (1/5 size of the full tablets cat. 11697498001) | Roche | 11836170001 | |
| MgCl2 | Ambion (Thermo) | AM9530G | |
| KCl | Ambion (Thermo) | AM9640G | |
| DTT | Thermo | R0861 | |
| Spermidine | Sigma | S2501 | |
| Spermine | Sigma | S3256-1G | |
| 70 um Flowmi cell strainers | Fisher | 03-421-228 | |
| 70 um bucket-style cell strainers | BD Falcon | 352350 | |
| Tris-HCl pH 7.5 | Invitrogen | 15567-027 | |
| NaCl | Ambion (Thermo) | AM9759 | |
| Tween 20 | Sigma | P2287-100ML | |
| H2O | Invitrogen | 10977-015 | |
| Dounce Tissue Grinder Set | Sigma | D8938-1SET | |
| INCYTO Disposable hemocytometers | Fisher | 22-600-100 | |
| BAM Banker | Wako Chemicals | 302-14681 | |
| RiboLock | Thermo | EO0384 | |
| 0.22 um PVDF Filter Units (500 ml) | Millipore | SCGVU05RE | |
| 0.22 um PVDF Filter Units (50 ml) | Millipore | SE1M179M6 | |
| Tn5 Transposase (TDE1) | Illumina | 15027865 | |
| 2x TD Tagment DNA Buffer | Illumina | 15027866 |
Before you start the protocol:
Before you start the protocol:
All steps should be performed on ice or at 4 °C . Pre-chill a fixed angle centrifuge to 4°C.
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
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.
Isolation of Nuclei via Bead Homogenization and Centrifuging
Isolation of Nuclei via Bead Homogenization and Centrifuging
move samples from liquid nitrogen, or -80 °C freezer onto On ice (wet 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.
if samples were slow-frozen in Bambanker, put the sample on On ice (wet ice) until the Bambanker thaws, then spin down sample 00:05:00 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.
5m
if samples are fresh, put the sample on on On ice (wet ice) until homogenization (200 µL pre-chilled HB should have been added to the tube before dissection.
Homogenize in BeadRupter Elite bead mill homogenizer using 2 cycles of the following:
Speed 1.0 for 00:00:20
Dwell for 00:00:20
Speed 2.0 for 00:00:20
Dwell for 00:00:20
1m 20s
Pellet nuclei by spinning 00:05:00 at 4 °C at 350 RCF in a fixed angle centrifuge.
5m
Re-suspend the nuclei in 1000 µL of ATAC-RSB-Tween Buffer
Centrifuge nuclei for 00:10:00 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.
Transposition of Nuclei
Transposition of Nuclei
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.
Add 50 ul ATAC-seq Reaction Mix to each tube and pipette up and down 6 times to resuspend nuclei pellet.
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.
Incubate reactions at 37 °C for 00:30:00 in a heat block (ideally should use a thermoshaker with 1000 RPM constant shaking).
After incubation, follow instructions of Qiage Minelute DNA clean-up Kit to purify the DNA
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
DNA Library Amplification
DNA Library Amplification
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.
Remove Hifi enzyme from freezer and place on wet ice. Make PCR master mix (MM)
PCR master mix (MM)
| A | B | C | D | |
| 1 | Reagent | Vol per sample 1x50 ul run (ul) | Vol (ul) made 2024.04.25/04.29 (10 + 1 samp.) | |
| 2 | H2O | 25.5 | 280.5 | |
| 3 | 2 X KAPA high fidelity butter | 10 | 110 | |
| 4 | dNTPs | 1.5 | 16.5 | |
| 5 | Hifi enzyme | 1 | 11 | |
| 6 | TOTAL MM VOLUME | 38 | 418 |
Mix and spin down master mix, keep cold, and add 38 uL to each reaction tube.
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.
primer plate in use:
| A | B | C | D | E | |
| 2024.04.29 | |||||
| plate number | 3 + 7 | H2O | 25.5 | 280.5 | |
| well number | 1-10 | 2 X KAPA high fidelity butter | 10 | 110 |
Add 2 uL of primer mix to each reaction. Make sure to record which index primers are used!
Add 10 uL of cleaned tagmentation product. Mix and spin down. Make sure to close PCR tube lids tightly with a marker end.
Place tubes in thermal cycler, making sure lid is tightly against your tubes, and run program ML2.
(Optional) Cleanup and Freezing Down of Extra Nuclei
(Optional) Cleanup and Freezing Down of Extra Nuclei
If you would like to save extra nuclei for other assays or to potentially use in additional ATAC-seq experiments downstream: