May 14, 2023
ATAC-seq from nuclei from frozen tissue
- 1Uppsala University
- methods
Protocol Citation: Eric RA Pederson 2023. ATAC-seq from nuclei from frozen tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6b4nkgqe/v1
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: Working
We use this protocol and it's working
Created: September 14, 2020
Last Modified: May 14, 2023
Protocol Integer ID: 42096
Keywords: ATAC-seq, frozen tissue, frozen tissue modified atac, seq method for frozen tissue, atac seq, seq from nuclei, atac, case brain tissue, seq method, seq, nuclei
Abstract
Modified ATAC-seq method for frozen tissue, in this case brain tissue.
Guidelines
The Tn5 enzyme used in this experiment was from a locally produced batch from the Protein Science Facility at the Karolinska Institutet in Stockholm, which is first discussed in Picelli and others (2014). Thus, if the Tn5 is purchased through a company it may react differently.
Materials
NEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541L
Digitonin, 40ulPromegaCatalog #G9441
Tn5 transposase with Nextera adapters loadedCatalog #UC-Macro-Tn5-Nextera adapter
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
Halt™ Protease Inhibitor Cocktail, EDTA-Free (100X)Thermo FisherCatalog #78437
SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOThermo FisherCatalog #S7563
Protease Inhibitor Tablets cOmplete Mini EDTA free RocheCatalog #11836170001
KIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938
2 ml LoBind TubesEppendorfCatalog #0030108078
1.5 mL LoBind tubes EppendorfCatalog #022431021
Falcon® 100 mm TC-treated Cell Culture DishCorningCatalog #353003
Equipment
DNA LoBind Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
0030108051
SKU
LINK
Qubit 2.0 Fluorometer
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Equipment
Countess 3 FL Automated Cell Counter
NAME
Automated Cell Counter
TYPE
Thermofisher scientific
BRAND
AMQAF2000
SKU
LINK
Equipment
Thermomixer C
NAME
Eppendorf
BRAND
2231000667
SKU
LINK
Equipment
Dounce, 2ml
NAME
Homogenizer
TYPE
KIMBLE
BRAND
432-0250
SKU
LINK
2 ml
SPECIFICATIONS
Equipment
Bio RS-24 Mini-rotator
NAME
mini-rotator
TYPE
BioSan
BRAND
RS-24
SKU
LINK
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
Mg(Ac)2
Tris-HCl pH 7.5
Sodium ChlorideFisher ScientificCatalog #S271
Magnesium ChlorideFisher ScientificCatalog #AC223210010
Molecular grade water nuclease-free
NN-Dimethylformamide (DMF) solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
Glycerol, 1000mlPromegaCatalog #H5433
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
| Amount | Reagents | |
| 3 ml | 1 M CaCl2 | |
| 0.6 ml | 3 M Mg(Ac)2 | |
| 6 ml | 1 M Tris pH 7.9 | |
| 89.2 ml | molecular grade water |
6x Homogenization Buffer Stable Solution.
Buffer recipes;
| A | B | |
| Amount | Reagents | |
| 2.27084 ml | 6x Homogenization Buffer stable | |
| 3.78 ml | 100mM PMSF | |
| 0.28 ul | 14.3 M B-mercaptoethanol | |
| 1/2 tablet | Protease inhibitor (cOmplete Mini) |
6x Homogenization Buffer Unstable Solution.
| Amount | Reagents | |
| 500ul | 1M Tris-Hcl ph 7.5 | |
| 100ul | 5M NaCl | |
| 150ul | 1M MgCl2 | |
| 49.25ml | H2O |
1x Homogenization Buffer Unstable Solution
| Amount | Reagents | |
| 500ul | 1M Tris-Hcl ph 7.5 | |
| 100ul | 5M NaCl | |
| 150ul | 1M MgCl2 | |
| 49.25ml | H2O |
ATAC-RSB
| Amount | Reagents | |
| 8 ml | ATAC-RSB | |
| 8 ul | 10% Tween |
ATAC-RSB + 10% Tween
2X TD buffer:
| Amount | Reagents | |
| 2 ml | 1 M Tris-Hcl pH 7.5 | |
| 1 ml | 1 M MgCl2 | |
| 20 ml | 100% Dimethyl Formamide |
2X TD buffer
(before the addition of dimethyl formamide, adjust the pH to 7.6 with 100% acetic acid)
DF buffer
| Amount | Reagents | |
| 100 mM | HEPES (pH 7.2) | |
| 200 mM | NaCl | |
| 0.2 mM | EDTA | |
| 2 mM | DTT | |
| 2.00% | Triton X-100 | |
| 20.00% | Glycerol |
2X DF buffer
| Primer | Sequence | Amount | |
| Tn5-A primer | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | 16 ul | |
| Tn5-rev primer | CTGTCTCTTATACACATCT | 16 ul |
Tube A
| Primer | Sequence | Amount | |
| Tn5-B primer | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG | 16 ul | |
| Tn5-rev primer | CTGTCTCTTATACACATCT | 16 ul |
Tube B
Protocol materials
Magnesium ChlorideFisher ScientificCatalog #AC223210010
NN-Dimethylformamide (DMF) solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551
Qubit 2.0 Fluorometer
Tn5 transposase with Nextera adapters loadedCatalog #UC-Macro-Tn5-Nextera adapter
Halt™ Protease Inhibitor Cocktail, EDTA-Free (100X)Thermo FisherCatalog #78437
Digitonin, 40ulPromegaCatalog #G9441
1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510
Molecular grade water nuclease-free
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
NEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541L
SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOThermo FisherCatalog #S7563
2 ml LoBind TubesEppendorfCatalog #0030108078
Falcon® 100 mm TC-treated Cell Culture DishCorningCatalog #353003
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Protease Inhibitor Tablets cOmplete Mini EDTA free RocheCatalog #11836170001
1.5 mL LoBind tubes EppendorfCatalog #022431021
Sodium ChlorideFisher ScientificCatalog #S271
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
Tris-HCl pH 7.5
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Glycerol, 1000mlPromegaCatalog #H5433
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
KIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938
Mg(Ac)2
50 ml Falcon tube
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
Safety warnings
Regular lab safety rules apply. Ensure the use of googles during the usage of dry ice.
Before start
The pre-experimentation steps are important
Also it is important that all the primers have been ordered and reconstituted beforehand.
Pre-experimentation
All steps should be performed on ice or at 4 °C .
1m
Pre-chill all Dounces and pestles to 4 °C in a fridge or on ice
10m
Pre-chill all tubes.
For each sample you are processing, you will need:
(i) One 2 mL 2 ml LoBind TubesEppendorfCatalog #0030108078 per sample
(ii) Three 1.5 mL LoBind tubes EppendorfCatalog #022431021 per sample
(iii) one PCR tube per sample
(iv) One 50 ml Falcon tube for filtration step per sample
10m
Prepare buffers.
i) 6x Homogenization Buffer Stable Solution.
ii) 6x Homogenization Buffer Unstable Solution.
iii) 1x Homogenization Buffer Unstable Solution.
iv) ATAC-RSB
v) ATAC-RSB + 10% Tween
vi) 2X TD buffer
vii) 2X DF buffer
1h 30m
i) 6x Homogenization Buffer stable Solution.
| A | B | |
| Amount | Reagents | |
| 3 ml | 1 M CaCl2 | |
| 0.6 ml | 3 M Mg(Ac)2 | |
| 6 ml | 1 M Tris pH 7.9 | |
| 89.2 ml | molecular grade water |
6x Homogenization Buffer Stable
20m
ii) 6x Homogenization Buffer Unstable Solution.
| Amount | Reagents | |
| 2.27084 ml | 6x Homogenization Buffer stable | |
| 3.78 ml | 100mM PMSF | |
| 0.28 ul | 14.3 M B-mercaptoethanol | |
| 1/2 tablet | Protease inhibitor (cOmplete Mini) |
6x Homogenization Buffer Unstable Solution
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
20m
ii) 1x Homogenization Buffer Unstable Solution.
| Amount | Reagents | |
| 2.166645 ml | 6x Homogenization Buffer Unstable Solution | |
| 4.16 ml | 1 M sucrose | |
| 2.6 ul | 500 mM EDTA | |
| 130 ul | 10.00% NP10 | |
| 6.540755 ml | H2O |
1x Homogenization Buffer Unstable Solution
20m
iii) ATAC-RSB:
| A | B | |
| Amount | Reagents | |
| 500ul | 1M Tris-Hcl ph 7.5 | |
| 100ul | 5M NaCl | |
| 150ul | 1M MgCl2 | |
| 49.25ml | H2O |
ATAC-RSB
20m
iv) ATAC-RSB + 10% Tween
| Amount | Reagents | |
| 8 ml | ATAC-RSB | |
| 8 ul | 10% Tween |
ATAC-RSB + 10% Tween
20m
v) 2X TD buffer
| A | B | |
| Amount | Reagents | |
| 2 ml | 1 M Tris-Hcl pH 7.5 | |
| 1 ml | 1 M MgCl2 | |
| 20 ml | 100% Dimethyl Formamide |
2X TD buffer
(before the addition of dimethyl formamide, adjust the pH to 7.6 with 100% acetic acid)
20m
vi) 2X DF buffer
| A | B | |
| Amount | Reagents | |
| 100 mM | HEPES (pH 7.2) | |
| 200 mM | NaCl | |
| 0.2 mM | EDTA | |
| 2 mM | DTT | |
| 2.00% | Triton X-100 | |
| 20.00% | Glycerol |
2X DF buffer
20m
Tn5 assembly reaction
2h
| A | B | C | |
| Primer | Sequence | Amount | |
| Tn5-A primer | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | 16 ul | |
| Tn5-rev primer | CTGTCTCTTATACACATCT | 16 ul |
Tube A
| A | B | C | |
| Primer | Sequence | Amount | |
| Tn5-B primer | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG | 16 ul | |
| Tn5-rev primer | CTGTCTCTTATACACATCT | 16 ul |
Tube B
95 °C to 25 °C
cooling -0.1°C/second
~1 hour on PCR machine
Olgio Annealing program
1h
TN5 Assembly
| A | B | |
| Amount | Reagent | |
| 25 ul | Tn5 | |
| 15.5 ul | Tube A | |
| 15.5 ul | Tube B | |
| 33 ul | 2X DF buffer |
Tn5 Assembly Mix
Room temperature 01:00:00
Tn5 from the Protein Science Facility at the Karolinska Institutet in Stockholm (Picelli et al.,2014)
or
Tn5 transposase with Nextera adapters loadedCatalog #UC-Macro-Tn5-Nextera adapter
1h
Nuclei extraction and filtration
Materials for the cutting of tissue on dry ice.
1m
- Gloves
- White warm gloves
- Dry ice
- Blades and handle
- Forceps
- Ethanol spray
- Cell culture dish
1m
In the most sterile way possible, cut a small piece of tissue, half a pea size or so, and leave it in the petri dish with a marking on the lid, in the dry ice. Weigh it and cut again if needed.
Make sure to use the ethanol to clean everything and be careful not to cut yourself.
20m
Add 2 mL 1X HB buffer into the dounce, which is sitting in the ice.
Add 0.2 µL 1 M DTT and and 1 µL 100X Halt protease inhibitor
2m
Place 20 mg frozen tissue into a pre-chilled 2 ml Dounce containing 1 ml cold 1x HB and let thaw for 00:05:00 .
5m
Dounce with “A” loose pestle until resistance goes away (~10 strokes).
Put the A pestle into the beaker of water
2m
Dounce with “B” tight pestle for 20 strokes.
Put the B pestle into the beaker of water
2m
Pour everything from the dounce into a 30 um MACS smartstrainer which is sitting on top of a labelled 50 ml falcon tube sitting in ice.
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
2m
Let it drip through for 10-15 minutes.
15m
Transfer to a labelled 2 mL Lobind Eppendorf tube, already cold from sitting in ice.
5m
To pellet the nuclei, centrifuge 900 rpm, 4°C, 00:10:00
10m
Transfer the supernatant to a new tube without disturbing the pellet
Repeat the centrifugation
10m
Discard supernatant
5m
Resuspend the nuclei in 200 µL of ATAC-RSB + 10% Tween
5m
Count the amount of cells using the cell counter in the cell lab.
20m
Put 10 µL Trypton blue onto a piece of parafilm and mix with 10 µL of the sample
Take 10 µL of the mix and pipette into a cell counting cell
Measure on the cell counting machine, making sure to adjust for the smaller size of the nuclei.
The machine will think they are dead cells.
Calculate the amount of nuclei to use for the next step;
(500000 nuclei)
5m
ATAC-seq requires 50,000 cells for the experiment. So for example if the number of nuclei is;
4.76 x 107
then the calculation will look like this;
50000 / 47600 = 1.10
Turn on the thermomixer to 37 °C
1m
ATAC-seq
27m
Add the calculated amount of cells into a2 mL lobind eppendorf tube with 1 ml ATAC-RSB + 10% Tween.
So taking the example above one would take 1.10 ul of nuclei in the 200 µL ATAC-RSB + 10% Tween to the new tube.
5m
Centrifuge nuclei for 00:10:00 900 rcf, 4°C, 00:10:00
10m
Here one can also save the nuclei for later. Simply spin the tube down with the new tube of diluted nuclei, discard the supernatant, add 300 µL of the and put in the -20° freezer.
00:10:00 900 rcf, 4°C, 00:10:00
10m
Discard most of the supernatant. Leave a bit in the bottom to ensure that there is enough for the reaction and that the pellet stays intact
2m
Add the reaction mix to the pellet and remaining water and resuspend the pellet in the mix
| A | B | |
| Amount | Reagents | |
| 25 ul | 2X TD buffer | |
| 16.5 ul | 1X PBS (cold) | |
| 0.5 ul | 1% digitonin | |
| 0.5 ul | 10% Tween -20 | |
| 2.5 ul | TN5 assembly |
Reaction Mix
5m
Put the tube into the thermomixer at 1000 rpm, 37°C, 00:30:00
30m
Take the tubes out and immediately proceed to the column clean up
2m
Use the Zymo DNA clean & concentrator to clean the nuclei
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
30m
Add 2-7 volumes of the DNA binding buffer to each volume of DNA sample
So in this case the volume is approximately 50 µL , so add 300 µL of DNA binding buffer
Transfer mixture to a provided Zymo-spin column in a collection tube
Centrifuge 10000 x g, 00:00:30
Discard supernatant
Add 200 µL DNA wash buffer to the column
Centrifuge 10000 x g, 00:00:30
repeat the wash step
Trasnfer the column to a 1.5 low-bind eppendorf tube.
Add 21 µL 2 DNA Elution buffer to the column.
Centrifuge 10000 x g, 00:00:30
Can stop here and start the next day if necessary.
Leave samples in 4°
Set up PCR;
| A | B | |
| Amount | Reagent | |
| 2.5 ul | Primer AD1 | |
| 2.5 ul | Primer AD2.# | |
| 25 ul | NEBNext Master Mix | |
| 20 ul | sample |
PCR
NEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541L
20m
PCR program - ATAC-seq pre-amplification
| A | B | C | |
| Tempurature | Time | Cycle | |
| 72° | 5 minutes | 1 | |
| 98° | 30 seconds | - | |
| 98° | 10 seconds | 5 | |
| 63° | 30 seconds | - | |
| 72° | 1 minute | - | |
| 72 | 5 minutes | 1 | |
| 4 | infinate | - |
ATAC-seq pre-amplification
40m
Remove PCR tubes from the machine and put immediately onto ice.
1m
Proceed immediately to the qPCR amplification to determine additional cycles step
1m
qPCR amplification to determine additional cycles
prepare a mix or master mix
| A | B | C | |
| Reagent | 1X | 6.5X | |
| Molecular grade water | 3.76 ul | 24.5 ul | |
| Primer AD1 | 0.5 ul | 6.5 ul | |
| Primer AD2.# | 0.5 ul | 0.5 ul * | |
| 25x SYBR green (in DMSO) | 0.24 ul | 1.56 ul | |
| 2x NEBNext Master Mix | 5 ul | 32.5 ul |
qPCR mix
SYBR™ Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOThermo FisherCatalog #S7563
NEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541L
1h 30m
qPCR program
| A | B | C | |
| Tempurature | Time | Cycle | |
| 98° | 30 seconds | 1 | |
| 98° | 10 seconds | 20 | |
| 63° | 30 seconds | - | |
| 72° | 1 minute | - |
qPCR program
qPCR extra setup options
- 6 machine not 6
- standard
- SYBR green
- no melt curve
- turn off ROX
- Fill in sample list
1m
Determine the required amount of cycles that each sample needs in addition
5m
Looking at the final amplification curve, determine the max fluorescence where the graph plateaus
Determine 1/3 of that number.
For example if the max is 1400000 then 1/3 would be 433333
Check the graph for how many cycles line up with this number from the curve.
In the case above it would be 6 because of where the line was
Continue the PCR with the additional amount of cycles skipping the beginning parts of the program
# of addtional cycles
| A | B | C | |
| Tempurature | Time | Cycle | |
| 98° | 10 seconds | Based on calculation | |
| 63° | 30 seconds | - | |
| 72° | 1 minute | - | |
| 4° | Infinate | 1 |
Additional cycles
40m
Take the tubes out and immediately proceed to the column clean up
1m
Use the Zymo DNA Clean & Concentrator to clean the nuclei
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
30m
Add 2-7 volumes of the DNA binding buffer to each volume of DNA sample
So in this case the volume is approximately 50 ul, so add 300 ul of DNA binding buffer
1m
Transfer mixture to a provided Zymo-spin column in a collection tube
1m
Centrifuge 10000 x g, 00:00:30
Discard supernatant
1m
Add 200 µL DNA wash buffer to the column
Centrifuge 10000 x g, 00:00:30
2m
Repeat the wash step
2m
Transfer the column to a 1.5 low-bind Eppendorf tube.
Add 21 ul Sterile water buffer to the column.
Centrifuge 10000 x g, 00:00:30
1m
Quality control
TapeStation (DS1000) and Qubit quantification
1h