ATAC-seq from nuclei from frozen tissue

Eric RA Pederson

May 14, 2023

ATAC-seq from nuclei from frozen tissue

DOI

dx.doi.org/10.17504/protocols.io.bp2l6b4nkgqe/v1

Eric RA Pederson¹

¹Uppsala University

Eric RA Pederson

Uppsala University

DOI: dx.doi.org/10.17504/protocols.io.bp2l6b4nkgqe/v1

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

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

ReagentNEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541L 
ReagentDigitonin, 40ulPromegaCatalog #G9441 
ReagentTn5 transposase with Nextera adapters loadedCatalog #UC-Macro-Tn5-Nextera adapter 
ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014 
ReagentMACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458 
ReagentHalt&trade; Protease Inhibitor Cocktail, EDTA-Free (100X)Thermo FisherCatalog #78437 
ReagentSYBR&trade; Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOThermo FisherCatalog #S7563 
ReagentProtease Inhibitor Tablets cOmplete Mini EDTA free RocheCatalog #11836170001 

ReagentKIMBLE 2mL Glass Dounce Tissue Grinder SetSigmaCatalog #D8938 
Reagent2 ml LoBind TubesEppendorfCatalog #0030108078  
Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021  
ReagentFalcon® 100 mm TC-treated Cell Culture DishCorningCatalog #353003 
 
Equipment
DNA LoBind Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
0030108051
SKU
https://online-shop.eppendorf.com/OC-en/Laboratory-Consumables-44512/Tubes-44515/DNA-LoBind-Tubes-PF-56252.html
LINK
ReagentQubit 2.0 Fluorometer   
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854  
Equipment
Countess 3 FL Automated Cell Counter
NAME
Automated Cell Counter
TYPE
Thermofisher scientific
BRAND
AMQAF2000
SKU
https://www.thermofisher.com/th/en/home/life-science/cell-analysis/cell-analysis-instruments/automated-cell-counters/models/countess-3-fl.html
LINK

Equipment
Thermomixer C
NAME
Eppendorf
BRAND
2231000667
SKU
https://www.pipette.com/2231000667-Promotion-Eppendorf-ThermoMixer-C-with-24x1-5-mL-SmartBlock-and-ThermoTop
LINK

Equipment
Dounce, 2ml
NAME
Homogenizer
TYPE
KIMBLE
BRAND
432-0250
SKU
https://se.vwr.com/store/product/561196/homogenisator-dounce-kimble
LINK
2 ml
SPECIFICATIONS

Equipment
Bio RS-24 Mini-rotator
NAME
mini-rotator
TYPE
BioSan
BRAND
RS-24
SKU
https://biosan.lv/products/-bio-rs-24-mini-rotator-for-test-tubes-with-timer/
LINK

Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
https://www.agilent.com/en/product/automated-electrophoresis/tapestation-systems/tapestation-instruments/4200-tapestation-system-228263
LINK
Reagent1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510  
 ReagentMg(Ac)2 
ReagentTris-HCl pH 7.5  
ReagentSodium ChlorideFisher ScientificCatalog #S271  
ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010  
ReagentMolecular grade water nuclease-free  
ReagentNN-Dimethylformamide (DMF) solutionMillipore SigmaCatalog #D4551  
ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML  
ReagentDTTSigma AldrichCatalog #D0632  
ReagentGlycerol, 1000mlPromegaCatalog #H5433  
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G  
Reagent2-MercaptoethanolSigma Aldrich  
ReagentHigh Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585  
ReagentHigh Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584  
 
AmountReagents
3 ml1 M CaCl2
0.6 ml3 M Mg(Ac)2
6 ml1 M Tris pH 7.9
89.2 mlmolecular grade water
6x Homogenization Buffer Stable Solution.
 

Buffer recipes;
AB
AmountReagents
2.27084 ml6x Homogenization Buffer stable
3.78 ml100mM PMSF
0.28 ul14.3 M B-mercaptoethanol
1/2 tabletProtease inhibitor (cOmplete Mini)
6x Homogenization Buffer Unstable Solution.

 
AmountReagents
500ul1M Tris-Hcl ph 7.5
100ul5M NaCl
150ul1M MgCl2
49.25mlH2O
1x Homogenization Buffer Unstable Solution
  


AmountReagents
500ul1M Tris-Hcl ph 7.5
100ul5M NaCl
150ul1M MgCl2
49.25mlH2O
ATAC-RSB
 
 
AmountReagents
8 mlATAC-RSB
8 ul10% Tween
ATAC-RSB + 10% Tween 
 



2X TD buffer:
 
AmountReagents
2 ml1 M Tris-Hcl pH 7.5
1 ml1 M MgCl2
20 ml100% Dimethyl Formamide
 2X TD buffer
 
(before the addition of dimethyl formamide, adjust the pH to 7.6 with 100% acetic acid)

DF buffer

 
AmountReagents
100 mMHEPES (pH 7.2)
200 mMNaCl
0.2 mMEDTA
2 mMDTT
2.00%Triton X-100
20.00%Glycerol
2X DF buffer
 

 
PrimerSequenceAmount
Tn5-A primerTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG16 ul
Tn5-rev primerCTGTCTCTTATACACATCT16 ul
Tube A
 
PrimerSequenceAmount
Tn5-B primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG16 ul
Tn5-rev primerCTGTCTCTTATACACATCT16 ul
Tube B
  

Protocol materials

ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632Materials
 
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854Materials
 
ReagentQubit 2.0 Fluorometer Materials
 
ReagentDigitonin, 40ulPromegaCatalog #G9441Materials
 
Reagent2 ml LoBind TubesEppendorfCatalog #0030108078Materials, Step 3
 
ReagentGlycerol, 1000mlPromegaCatalog #H5433Materials
 
ReagentMACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458Materials, Step 12
 
ReagentTn5 transposase with Nextera adapters loadedCatalog #UC-Macro-Tn5-Nextera adapterMaterials, Step 5.2
 
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260GMaterials
 
ReagentSYBR&trade; Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOThermo FisherCatalog #S7563Materials, Step 32
 
Reagent1.5 mL LoBind tubes EppendorfCatalog #022431021Materials, Step 3
 
ReagentKIMBLE 2mL Glass Dounce Tissue Grinder SetMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8938Materials
 
ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010Materials
 
ReagentMolecular grade water nuclease-freeMaterials
 
ReagentNN-Dimethylformamide (DMF) solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551Materials
 
ReagentHalt&trade; Protease Inhibitor Cocktail, EDTA-Free (100X)Thermo FisherCatalog #78437Materials
 
Reagent1 M Calcium Chloride (CaCl2)Fisher ScientificCatalog #BP510Materials
 
ReagentFalcon® 100 mm TC-treated Cell Culture DishCorningCatalog #353003Materials
 
ReagentTris-HCl pH 7.5Materials
 
ReagentMg(Ac)2Materials
 
ReagentProtease Inhibitor Tablets cOmplete Mini EDTA free RocheCatalog #11836170001Materials
 
ReagentSodium ChlorideFisher ScientificCatalog #S271Materials
 
ReagentHigh Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584Materials
 
Reagent2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Materials
 
ReagentHigh Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585Materials
 
ReagentNEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsNew England BiolabsCatalog #M0541LIn Materials and 2 steps
 
ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014In Materials and 2 steps
 
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50MLMaterials
 
Reagent50 ml Falcon tubeStep 3
 
ReagentcOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)Step 4.2

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 Temperature4 °C  . 

Pre-chill all Dounces and pestles to Temperature4 °C   in a fridge or on ice

10m

Pre-chill all tubes.

For each sample you are processing, you will need: 
(i) One Amount2 mL    Reagent2 ml LoBind TubesVWR InternationalCatalog #0030108078  per sample
(ii) Three Reagent1.5 mL LoBind tubes VWR InternationalCatalog #022431021  per sample
(iii) one PCR tube per sample 
(iv) One Reagent50 ml Falcon tubeVWR International  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.
 
AB
AmountReagents
3 ml1 M CaCl2
0.6 ml3 M Mg(Ac)2
6 ml1 M Tris pH 7.9
89.2 mlmolecular grade water
 6x Homogenization Buffer Stable

20m

ii) 6x Homogenization Buffer Unstable Solution.
 
AmountReagents
2.27084 ml6x Homogenization Buffer stable
3.78 ml100mM PMSF
0.28 ul14.3 M B-mercaptoethanol
1/2 tabletProtease inhibitor (cOmplete Mini)
 6x Homogenization Buffer Unstable Solution
 
ReagentcOmplete™, Mini, EDTA-free (Protease Inhibitor)VWR InternationalCatalog ##11836170001)  

20m

ii) 1x Homogenization Buffer Unstable Solution.
 
AmountReagents
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 mlH2O
1x Homogenization Buffer Unstable Solution

20m

iii) ATAC-RSB: 
AB
AmountReagents
500ul1M Tris-Hcl ph 7.5
100ul5M NaCl
150ul1M MgCl2
49.25mlH2O
ATAC-RSB
 

20m

iv)  ATAC-RSB + 10% Tween  
AmountReagents
8 mlATAC-RSB
8 ul10% Tween
ATAC-RSB + 10% Tween 
 

20m

v) 2X TD buffer
 
AB
AmountReagents
2 ml1 M Tris-Hcl pH 7.5
1 ml1 M MgCl2
20 ml100% 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

 
AB
AmountReagents
100 mMHEPES (pH 7.2)
200 mMNaCl
0.2 mMEDTA
2 mMDTT
2.00%Triton X-100
20.00%Glycerol
2X DF buffer
 

20m

Tn5 assembly reaction

ABC
PrimerSequenceAmount
Tn5-A primerTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG16 ul
Tn5-rev primerCTGTCTCTTATACACATCT16 ul
Tube A


ABC
PrimerSequenceAmount
Tn5-B primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG16 ul
Tn5-rev primerCTGTCTCTTATACACATCT16 ul
Tube B

Temperature95 °C   to Temperature25 °C   
cooling -0.1°C/second

~1 hour on PCR machine
Olgio Annealing program

TN5 Assembly


AB
AmountReagent
25 ulTn5
15.5 ulTube A
15.5 ulTube B
33 ul2X DF buffer
Tn5 Assembly Mix

TemperatureRoom temperature   Duration01:00:00   


Tn5 from the Protein Science Facility at the Karolinska Institutet in Stockholm (Picelli et al.,2014)
or
ReagentTn5 transposase with Nextera adapters loadedVWR InternationalCatalog #UC-Macro-Tn5-Nextera adapter 

Nuclei extraction and filtration

Materials for the cutting of tissue on dry ice.

- Gloves
- White warm gloves
- Dry ice
- Blades and handle
- Forceps
- Ethanol spray
- Cell culture dish 

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 Amount2 mL   1X HB buffer into the dounce, which is sitting in the ice.

Add  Amount0.2 µL   1 M DTT and and Amount1 µL   100X Halt protease inhibitor 

Place 20 mg frozen tissue into a pre-chilled 2 ml Dounce containing 1 ml cold 1x HB and let thaw for Duration00:05:00  . 

Dounce with “A” loose pestle until resistance goes away (~10 strokes).

Put the A pestle into the beaker of water

Dounce with “B” tight pestle for 20 strokes.

Put the B pestle into the beaker of water

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.
ReagentMACS SmartStrainers 30umVWR InternationalCatalog #130-098-458 

Let it drip through for 10-15 minutes.

15m

Transfer to a labelled Amount2 mL   Lobind Eppendorf tube, already cold from sitting in ice.

To pellet the nuclei, centrifuge Centrifigation900 rpm, 4°C, 00:10:00  

10m

Transfer the supernatant to a new tube without disturbing the pellet

Repeat the centrifugation

10m

Discard supernatant

Resuspend the nuclei in Amount200 µL   of ATAC-RSB + 10% Tween

Count the amount of cells using the cell counter in the cell lab.

20m

Put Amount10 µL   Trypton blue onto a piece of parafilm and mix with Amount10 µL   of the sample

Take Amount10 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)

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 Temperature37 °C   

ATAC-seq

27m

Add the calculated amount of cells into aAmount2 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 Amount200 µL   ATAC-RSB + 10% Tween to the new tube.

Centrifuge nuclei for Duration00:10:00   Centrifigation900 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 Amount300 µL    of the and put in the -20° freezer.

 Duration00:10:00   Centrifigation900 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

Add the reaction mix to the pellet and remaining water and resuspend the pellet in the mix


AB
AmountReagents
25 ul2X TD buffer
16.5 ul1X PBS (cold)
0.5 ul1% digitonin
0.5 ul10% Tween -20
2.5 ulTN5 assembly
Reaction Mix

Put the tube into the thermomixer at Shaker1000 rpm, 37°C, 00:30:00 

30m

Take the tubes out and immediately proceed to the column clean up

Use the Zymo DNA clean & concentrator to clean the nuclei
ReagentZymo DNA Clean & Concentrator - 5VWR InternationalCatalog #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 Amount50 µL   , so add Amount300 µL   of DNA binding buffer

Transfer mixture to a provided Zymo-spin column in a collection tube

Centrifuge Centrifigation10000 x g, 00:00:30  
Discard supernatant

Add Amount200 µL   DNA wash buffer to the column

Centrifuge Centrifigation10000 x g, 00:00:30  

repeat the wash step

Trasnfer the column to a 1.5 low-bind eppendorf tube.

Add Amount21 µL   2 DNA Elution buffer to the column.

Centrifuge Centrifigation10000 x g, 00:00:30  

Can stop here and start the next day if necessary.
Leave samples in 4°

Set up PCR;


AB
AmountReagent
2.5 ulPrimer AD1
2.5 ulPrimer AD2.#
25 ulNEBNext Master Mix
20 ulsample
PCR


ReagentNEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsVWR InternationalCatalog #M0541L 

20m

PCR program - ATAC-seq pre-amplification

ABC
TempuratureTimeCycle
72°5 minutes1
98°30 seconds-
98°10 seconds5
63°30 seconds-
72°1 minute-
725 minutes1
4infinate-
ATAC-seq pre-amplification

40m

Remove PCR tubes from the machine and put immediately onto ice.

Proceed immediately to the qPCR amplification to determine additional cycles step

qPCR amplification to determine additional cycles

prepare a mix or master mix


ABC
Reagent1X6.5X
Molecular grade water3.76 ul24.5 ul
Primer AD10.5 ul6.5 ul
Primer AD2.#0.5 ul0.5 ul *
25x SYBR green (in DMSO)0.24 ul1.56 ul
2x NEBNext Master Mix5 ul32.5 ul
qPCR mix


ReagentSYBR&trade; Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSOVWR InternationalCatalog #S7563 

ReagentNEBNext High-Fidelity 2X PCR Master Mix - 250 rxnsVWR InternationalCatalog #M0541L 

1h 30m

qPCR program


ABC
TempuratureTimeCycle
98°30 seconds1
98°10 seconds20
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

Determine the required amount of cycles that each sample needs in addition

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

ABC
TempuratureTimeCycle
98°10 secondsBased on calculation
63°30 seconds-
72°1 minute-
4°Infinate1
Additional cycles

40m

Take the tubes out and immediately proceed to the column clean up

Use the Zymo DNA Clean & Concentrator to clean the nuclei

ReagentZymo DNA Clean & Concentrator - 5VWR InternationalCatalog #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

Transfer mixture to a provided Zymo-spin column in a collection tube

Centrifuge Centrifigation10000 x g, 00:00:30  
Discard supernatant

Add Amount200 µL   DNA wash buffer to the column

Centrifuge Centrifigation10000 x g, 00:00:30  

Repeat the wash step

Transfer the column to a 1.5 low-bind Eppendorf tube.

Add 21 ul Sterile water buffer to the column.

Centrifuge Centrifigation10000 x g, 00:00:30  

Quality control
TapeStation (DS1000) and Qubit quantification


	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

	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)


	Amount	Reagents
	500ul	1M Tris-Hcl ph 7.5
	100ul	5M NaCl
	150ul	1M MgCl2
	49.25ml	H2O


	Amount	Reagents
	2 ml	1 M Tris-Hcl pH 7.5
	1 ml	1 M MgCl2
	20 ml	100% Dimethyl Formamide


	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


Primer	Sequence	Amount
Tn5-A primer	TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG	16 ul
Tn5-rev primer	CTGTCTCTTATACACATCT	16 ul


	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

	A	B
	Amount	Reagent
	25 ul	Tn5
	15.5 ul	Tube A
	15.5 ul	Tube B
	33 ul	2X DF buffer

	A	B
	Amount	Reagent
	2.5 ul	Primer AD1
	2.5 ul	Primer AD2.#
	25 ul	NEBNext Master Mix
	20 ul	sample

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	-

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

Public workspaceATAC-seq from nuclei from frozen tissue

ATAC-seq from nuclei from frozen tissue