Nov 27, 2019

Public workspace3XFlag-pATn5 Protein Purification and MEDS-loading (5x scale, 2L volume)

eLife
DOI
dx.doi.org/10.17504/protocols.io.8yrhxv6
  • 1Fred Hutchinson Cancer Research Center, HHMI;
  • 2Fred Hutchinson Cancer Research Center
Terri Bryson
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DOI: dx.doi.org/10.17504/protocols.io.8yrhxv6
External link: https://www.nature.com/articles/s41467-019-09982-5
Protocol Citation: Terri Bryson, Steven Henikoff 2019. 3XFlag-pATn5 Protein Purification and MEDS-loading (5x scale, 2L volume). protocols.io https://dx.doi.org/10.17504/protocols.io.8yrhxv6
Manuscript citation:
Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S: CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature communications 2019, In press.
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: November 03, 2019
Last Modified: November 27, 2019
Protocol Integer ID: 29425
Keywords: CUT&Tag, pATn5, pATn5+MEDS
Abstract
Here we describe the method used to purify and load pATn5 for CUT&Tag, https://www.protocols.io/view/bench-top-cut-amp-tag-z6hf9b6/abstract. This protocol was modeled after that in Picelli et al (Picelli et al., 2014) using the pTXB1-rbs_3XFlag-pATn5-FL plasmid available from Addgene: https://www.addgene.org/124601/. It yields, as written, Amount8.7 mL loaded pATn5 used at 1:200 dilution in CUT&Tag reactions, and Amount10.5 mL unloaded pATn5 for future use, both of which should be stored at -20C and can easily be scaled down.

Materials
Equipment
  • RC5C Sorvall centrifuge or similar
  • Branson Sonifier 250 fitted with the Branson Ultrasonics™ Sonifier™ Micro-tip
  • Eppendorf 5804R, or similar


Reagents
  • Ampicillin, 100mg/mL stock: Sigma-Aldrich A9518
  • Carbenicillin, 100mg/mL stock: Disodium Salt Thermo Fisher Scientific cat# 10177012,
  • LB: Miller’s LB Broth, Corning Cat# 46050CM
  • IPTG, 1M Stock: ISOPROPYL B-D-THIOGALACTOPYRANOSIDE, Sigma I6758-1G
  • DTT, 1M stock: Sigma D0632
  • Roche Complete EDTA free protease inhibitor (PI) tablets, Sigma 5056489001
  • Chitin Resin (NEB S6651S)

Consumables:
  • Econo-Pac® Chromatography Columns, (BIO-RAD 732-1010)
  • 50 mL Conical Tubes, Fisher 14-959-49A
  • 4x Millipore Centrifugal Filter Units 30K, Amicon Ultra-15 Cat# UFC903024
  • Slide A Lyzer 10K MWCO Dialysis Cassettes, Fisher PI66830
  • 18G needles
  • foam buoy
  • Zip Tie 1
  • ¼ inch binder clip
  • 2-inch stirbar

Buffers and Solutions


1L HEGX Buffer: add Protease Inhibitor Tablets just before use. Sigma #5056489001
1L HEGX Buffer:Vol (mL)Stock Solution
0.02 M HEPES-KOH at pH 7.2201M
1 M NaCl2005M
0.001 M EDTA 20.5M
10% glycerol100100%
0.2% Triton X-100 2100%
676 mL Water

Tn5 Dialysis Buffer 5 L total (make 2x 2.5L in 4 L beakers)
2X Tn5 Dialysis Buffer: 1.6L totalVol (mL)Stock Solution
0.1M HEPES-KOH at pH 7.2 1601M
0.2 M NaCl645M
0.2 mM EDTA0.640.5M
1.7 mM DTT2.721M
0.2% Triton X-1003.2100%
20% glycerol 320100%
1049.44 mL Water


1M HEPES-KOH pH 7.2
  • 400mL Water
  • 119g HEPES
  • ~7.12g KOH pellets to pH7.2
  • Adjust water to 500mL
  • Filter Sterilize
  • Store at Temperature4 °C

MEDS Assembly and In Vitro Assay
  • Annealing Buffer (10mM Tris pH 8, 50mM NaCl, 1mM EDTA).
  • MED Oligos: Eurofins, salt-free purification (need reference for sequences here)
  • Glycerol, Sigma G5516
  • 150ng HMW Lambda DNA (NEB# N3011)
  • Proteinase K, Thermo Fisher Scientific 25530049
  • SDS, Sigma L4509
  • 1KB+ MW Marker, Fermentas SM1343
  • HMW Lambda DNA, NEB# N301
  • EtBr, Invitrogen 15585011
  • Agarose, Thermo Fisher Scientific 16500500

MEDS Analysis: 6% Acrylamide Gel for DNA- 1xTBE gel, run in 1xTBE buffer
  • 1.35 mL 40% Acrylamide 19:1
  • 90 uL 10% APS (freshly made)
  • 13.5uL TEMED
  • 0.9mL 10x TBE
  • 6.65mL Water
  • Alternatively, you can purchase a premade gel from Invitrogen: https://www.thermofisher.com/order/catalog/product/EC62652BOX?SID=srch-srp-EC62652BOX
  • 6X Fermentas Orange Loading Dye, Thermo Fisher Scientific R0631
  • 10X TBE, Fisher BP1333-1

5XTAPS-MgCl2-PEG8000
  • 50 mM TAPS-NaOH pH8.5, Millipore Sigma T5130-25G (From 1M TAPS, pH to 8.5 with NaOH)
  • 25 mM MgCl2
  • 40% PEG 8000 (Fisher 202452)

Protein Analysis

3x SDS sample buffer
http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10075.full?sid=4f034b3d-e1db-4b81-b971-87014561be54
  • 188 mM Tris-Cl (pH 6.8)
  • 3% SDS
  • 30% glycerol
  • 0.01% bromophenol blue
  • 15% β-mercaptoethanol

Coomassie brilliant blue R250 (0.1% w/v)
http://cshprotocols.cshlp.org/content/2007/2/pdb.rec10716.full?sid=92093ab7-2ae7-4b2e-90ab-f676ab3ba5de
  • Coomassie brilliant blue R250
  • Methanol
  • Acetic acid
Prepare the Coomassie brilliant blue R250 in a 60/30/10 (v/v/v) mixture of H2O, methanol, and acetic acid.

Destaining solution for Coomassie brilliant blue R250
http://cshprotocols.cshlp.org/content/2007/2/pdb.rec10717.full?sid=98f359ff-80c0-4da8-8c24-f6c4bfa5a66e
  • Methanol
  • Acetic acid
Mix H2O, methanol, and acetic acid in a ratio of 50/40/10 (v/v/v).


Material for PAGE Analysis of pATn5 purification:


Reagents for testing transposase activity in vitro:
  • loaded and unloaded pATn5 in 50% glycerol
  • High molecular weight DNA (NEB #N3011 Lambda DNA) diluted 1:10 from 500ng/uL to 50ng/uL
  • 5XTAPS-MgCl2-PEG8000
  • 1% SDS
  • Proteinase K
  • 0.7% Agarose gel made with 1x TAE and running buffer


Safety warnings
For safety warnings and hazard information, please refer to the SDS (Safety Data Sheet).


Day 1
Day 1
15m
15m
Streak E. coli glycerol stock containing pTXB1-rbs_3XFlag-pATn5-FL plasmid onto an LB-Amp100 plate, incubate DurationOvernight at Temperature37 °C

Day 2
Day 2
2h
2h
Select a single colony and make a patch plate for later, use within 1 week.

Make buffers and media.
Note

Day 3: Culture and Induce
Day 3: Culture and Induce
8h
8h
Early morning, with a sterile toothpick swipe through a bacterial patch to inoculate Amount30 mL of LB broth+ carbenicillin 100ug/mL culture, let shake ~ Duration04:00:00 .
Put Amount1970 mL of LB+ carbenicillin 100ug/mL in 6L flask at Temperature37 °C as well so it is already warm at the time of dilution.
Note
Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so it is potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, it is also more expensive.

After Duration04:00:00 incubation use all 30 ml of starter culture to inoculate larger vessel.

Shake at Temperature37 °C , check OD600 periodically until it reaches O.D600 of 0.5-0.7. ~Duration03:00:00

Move to cold room to let cool ~Duration01:00:00

Induce culture with addition of fresh 1M IPTG to 0.25mM final and incubate on the Temperature18 °C shaker ~DurationOvernight

Day 4: Pellet and Freeze
Day 4: Pellet and Freeze
2h
2h
Pellet bacteria by centrifugation: Divide the culture between 6x Amount500 mL bottles, centrifuge at 9500rcf, 4C for Duration01:00:00 in a GS3 Rotor, RC5C Sorvall centrifuge.

2h
Discard the supernatant.
Freeze bacterial pellets in dry ice and ethanol bath.
Store at Temperature-80 °C freezer until ready to process.

Day 5: Lyse, Chitin Binding
Day 5: Lyse, Chitin Binding
4h
4h

Note
Need Amount400 mL HEGX+PI today; make a total of 1L HEGX and add PI tablets as needed each day.








Remove pellets from freezer, thaw on ice and resuspend together in a combined total of Amount200 mL of chilled HEGX Buffer with Roche Complete EDTA free protease inhibitor (PI) tablets. Keep on ice while working.

Divide combined resuspension between 4x clean 100 ml plastic beakers nested in a larger plastic beaker filled with ice. See figure A.



While keeping in the ice nest, sonicate each sample 10-12 times, Duration00:00:45 each at 50% duty cycle and output level 7 on a Branson Sonifier 250 fitted with the Branson Ultrasonics™ Sonifier™ micro-tip. Between rounds of sonication keep in larger ice bucket (see figure A) and use a 2mL serological pipette to gently stir to facilitate cooling. Let each sample rest at least Duration00:00:45 between sonication rounds.

Safety information
To prevent foaming keep the sonicator tip in the center of the sample. If excessive foaming occurs during sonication either wait for foam to settle or remove with a pipette and discard.



Transfer to fresh 50 mL conical tubes and pellet the lysate by centrifugation for Duration00:30:00 , Temperature4 °C , at Centrifigation16000 x g .


Safety information
Chitin Binding: The remainder of the protocol is carried out at Temperature4 °C or on ice. All buffers should be chilled on ice prior to use.


In Temperature4 °C room prepare 10x Econo-Pac® Chromatography Columns (BIO-RAD 732-1010), each holds Amount20 mL Set-up columns (we use a rack intended for Qiagen Maxi-preps, figure B). Gently invert Chitin Resin (NEB S6651S) to fully suspend and add 2.5mL to each column. Wash each with Amount20 mL of HEGX Buffer, drain immediately, don’t collect anything. Do not let resin dry out prior to addition of sample.


Add yellow cap to bottom of column.
Add supernatant from lysate spin to Chitin Resin slowly to prevent bubbles.
Cap column tightly and incubate DurationOvernight at Temperature4 °C on nutator. Check for leakage after Duration00:15:00 andDuration00:30:00 , wrap both ends in parafilm if necessary to prevent leakage.

Day 6: Wash and Cleavage Intein with DTT.
Day 6: Wash and Cleavage Intein with DTT.
1h
1h
Return columns to rack: remove yellow cap gently, hang rack over collection tube and uncap column. Drain unbound fraction into 50 mL tubes (save on ice or at Temperature4 °C in the event troubleshooting is required later).
Wash column 2x with Amount20 mL HEGX collecting first wash into 50 mL tubes (save on ice or at Temperature4 °C in the event troubleshooting is required later), the second wash can be wasted.

Add yellow cap to bottom of column.
Make Amount65 mL HEGX +PI with 100mM DTT and slowly add Amount6 mL to each sample.

Cap column tightly and nutate in cold room ~Duration48:00:00

Day 7: Elute & Dialyze
Day 7: Elute & Dialyze
4h
4h
Return columns to rack: remove yellow cap gently, hang in rack over collection tube and uncap column. Drain unbound fraction into 15 mL conical tubes, combine at end. Cover resin with additional Amount2 mL of buffer and save at Temperature4 °C until purification is verified.

Record elution volume. ~Amount50 mL

Dialyze the protein as described in the following substeps.
Assemble 2 Slide-A-Lyzer 10K MWCO Dialysis Cassettes
  • Use a medium (1 ¼ inch) binder clip to secure a 2 inch stir bar and remove the clip handles gently. Use an 18-inch zip tie to secure a foam float to the cassette. See figure C.
  • Hydrate the cassette by submerging and use a binder clip to anchor the cassette at the proper depth by clipping the zip tie tail to the lip of the dialysis beaker. Let hydrate according to product instructions


Using an 18 gauge needle load the Slide-A-Lyzer Cassettes with Amount25 mL each. Follow product literature on how to properly load cassettes.
In a 4 L beaker, add both cassettes and dialyze for Duration01:00:00 -Duration02:00:00 in Amount2.5 L of Tn5 dialysis buffer with DTT added to 1.7mM just before use.
Move samples to fresh Tn5 dialysis buffer, also Amount2.5 L with DTT added to 1.7mM just before use, let dialyze DurationOvernight at Temperature4 °C .
Recover sample from cassette and combine into a fresh 50mL conical tube. Hold on ice or at Temperature4 °C until next steps. Overnight storage is ok if this falls into a weekend.
Note
Volume recovered from Dialysis: ~Amount42 mL


Day 8: Concentrate Protein
Day 8: Concentrate Protein
1h 30m
1h 30m
Separate dialyzed protein between 4x Millipore Centrifugal Filter Units 30K, Amicon Ultra-15 Cat# UFC903024
Spin in Eppendorf 5804R at Centrifigation2880 x g and Temperature4 °C for Duration01:00:00 . Check at Duration00:20:00 intervals to assess progress, do not let volume drop below top of filters.

Recover concentrated sample from the upper filter portion of the tube, use an extra 500uL of buffer from lower catch tube to wash any extra protein off of each filter and add to collected protein.
Note
Volume recovered from Concentrator: 7 mL + 2mL for rinsing filter = 9 mL

Add an equal volume of sterile 80% glycerol to the concentrated protein. Combined with the glycerol from dialysis the final glycerol concentration is ~50%.
Store at Temperature-20 °C .

PAGE Analysis of pATn5 purification
PAGE Analysis of pATn5 purification

Note
Material:
• Novex; 4-20% Tris-Glycine Gel 1.0 mm, 12 well WEDGE, Thermo Fisher Scientific cat# XP04120PK2
• Pierce™ Bovine Serum Albumin Standard Pre-Diluted Set, Thermo Fisher Scientific cat# 23208
• PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, Thermo Fisher Scientific cat 26620
• 2-Mercaptoethanol, BIO-RAD cat#1610710
• 1xTris Glycine Buffer



Run small volume of purified protein (and some from a previous batch if available) to compare yields. Include BSA standard range for quantification if desired. To the 3x Loading Dye from CSH Protocols, add 2-mercaptoethanol to 15% final before adding to the protein samples, heat to Temperature100 °C for Duration00:10:00 cool back to TemperatureRoom temperature prior to loading onto gel.

To make the BSA Standards dilute the 250 ng/uL BSA to 125 ng/uL and use increasing volumes on the gel.
Sample Prep and Gel Loading (figure D)

Lanes L→RVolume of protein, uL3xLoading Dye (+2-ME)1xTE
Protein Marker6--
125ng BSA1310
250ng BSA239
500ng BSA438
750ng BSA637
1.5ug BSA123-
Protein Marker6--
pATn5 -Batch 21.5310
pATn5 -Batch 31.5310
pATn5 -Batch 33.038


After loading samples run gel at 30mA for ~Duration01:00:00 or until the loading dye leading edge runs to the bottom of the gel.

Transfer gel carefully to covered dish with Coomassie stain gently shake at least Duration01:00:00 to DurationOvernight at TemperatureRoom temperature .

Replace stain with destain, add a rolled paper towel to dish to absorb excess Coomassie dye.
Replace destain with water, scan gel for notes.
Annealing of MEDS Oligos
Annealing of MEDS Oligos


Tube #Seq NameSeq 5' to 3'ODVol. µlnmolµgLenMW% GC ContentE260TmScalePurif.Vol in µl for 100 µMVol in µl for 200 µM
1TN5MErev[Phos]CTGTCTCTTATACACATCT30dry178.81031.5195768.836.816778353.71.0 µmolSalt-Free1788894
2Tn5ME-ATCGTCGGCAGCGTCAGATGTGTATAAG AGACAG161.5dry484.24959.63310242.851.533345670.91.0 µmolSalt-Free48421210.5
3Tn5ME-BGTCTCGTGGGCTCGGAGATGTGTATAA GAGACAG188.3dry550.75839.5341060352.934195671.91.0 µmolSalt-Free55072753.5
Data Sheet from Eurofins

Resuspend single-stranded salt-free oligos from Eurofins to 200uM in Annealing Buffer (10mM Tris Ph8 , 50mM NaCl, 1mM EDTA).
Mix equal volumes of both complementary oligos (at equimolar concentration) in a Amount1.5 mL microfuge tube, mix well and aliquot to in smaller tubes appropriate for thermal cycler.

Amount400 µL Tn5MErev + Amount400 µL Tn5ME-A = Amount800 µL → 4 tubes @ Amount200 µL
Amount400 µL Tn5MErev + Amount400 µL Tn5ME-B = Amount800 µL → 4 tubes @ Amount200 µL
In MJ Cycler PTC-200 use ANNEALOL program which starts with a Temperature95 °C Duration00:02:00 incubation and decreases temp in Temperature5 °C increments incubating Duration00:05:00 each ending with a Duration00:05:00 incubation at Temperature25 °C and holds at Temperature8 °C afterwards. Store at Temperature-20 °C .
Verify annealing:
Run oligos, both ss and ds, on a 6% acrylamide 1xTBE (figure E) Lanes:

1. 1KB+ Marker 3uL
2. Rev+ ME-A 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE
3. Rev+ ME-B 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE
4. Rev 1uL +1uL 6X Fermentas Orange Loading Dye +6uL TE
5. ME-A 0.5uL +1uL 6X Fermentas Orange Loading Dye +6uL TE
6. ME-B 0.5uL +1uL 6X Fermentas Orange Loading Dye +6uL TE
7. 100 bp Marker 3uL
Note
While annealing efficiency is not 100% it is robust, excess ssDNA will not load onto transposase and will wash away during CUT&Tag protocol.


Assembly of Tn5 with pre-annealed MEDS and in vitro assay to verify tagmentation activity
Assembly of Tn5 with pre-annealed MEDS and in vitro assay to verify tagmentation activity
With oligo-mer:

  • Amount600 µL 100 uM Tn5ME-rev/A oligo
  • Amount600 µL 100 uM Tn5ME-rev/B oligo
  • Amount7.5 mL 3xFlag-pATn5 (in 50% glycerol)

The mixture is incubated for ~Duration00:50:00 at TemperatureRoom temperature and then stored at Temperature-20 °C .
Testing transposase activity in vitro
Testing transposase activity in vitro
In vitro activity assay is performed on 150 ng HMW Lambda DNA (NEB# N3011). Unloaded pATn5 or loaded pATN5 without MgCl2 should have no impact on HMW DNA, loaded pATN5 in digestion buffer should digest HMW DNA to a smear. See figure F.
For each sample prepare the following reaction mix:
  • Amount150 ng HMW DNA
  • Amount4 µL 5XTAPS-MgCl2-PEG8000 (water for no digestion buffer controls)
  • Amount5 µL pATn5-MEDS complex
  • To 20ul Nuclease-Free H2O
Incubate for Duration00:07:00 at Temperature55 °C .
To release and inactivate Tn5, add Amount2 µL of 1%SDS (disrupt protein DNA interaction).
Incubate for Duration00:07:00 at TemperatureRoom temperature or Temperature55 °C .
Add Amount0.63 µL proteinase K (20 mg/mL) to each reaction (degrade pATn5).
Incubate for Duration00:07:00 at Temperature55 °C .
Keep TemperatureOn ice or Temperature4 °C .
The samples can be analyzed by migration on 0.7% agarose gel in 1xTAE.
Expected result
Gel Lanes: (figure F)

1. 150ng HMW lambda DNA
2. 1KB+ MW Marker
3. pATn5 (-) MEDS
4. pATn5 (+) MEDS, (-) digestion buffer
5. pATn5 (+) MEDS, (+) digestion buffer
6. pATn5[batch 2], (+) MEDS, (+) digestion buffer