Nov 27, 2024

Public workspaceIn vivo tissue-specific chromatin profiling in Drosophila V.3

DOI
dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v3
  • 1Purdue University
  • WeakeLab
Vikki M. Weake
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DOI: dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v3
External link: https://doi.org/10.1093/genetics/iyab079
Protocol Citation: Vikki M. Weake, Juan P P Jauregui-Lozano, Sarah E McGovern, Gaoya Meng, Makayla N Marlin 2024. In vivo tissue-specific chromatin profiling in Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v3Version created by Vikki M. Weake
Manuscript citation:
Juan Jauregui-Lozano, Kimaya Bakhle, Vikki M Weake, In vivo tissue-specific chromatin profiling in Drosophila melanogaster using GFP-tagged nuclei, Genetics, Volume 218, Issue 3, July 2021, iyab079, https://doi.org/10.1093/genetics/iyab079
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, 2023
Last Modified: November 27, 2024
Protocol Integer ID: 112731
Abstract
Chromatin regulation plays an essential role in many nuclear processes and genome-wide chromatin profiling approaches contribute to understanding how chromatin regulates cell homeostasis. Chromatin dysregulation lies at the heart of many human diseases, most of which have a tissue-specific nature. Because of the physiological similarity of Drosophila and humans, tissue-specific studies can be performed using fruit flies. Here, we present an improved nuclear tagging approach that allows for efficient purification of cell-type specific nuclei from Drosophila increasing yield and stringency. Using this protocol, we purified photoreceptor neuron nuclei and demonstrate the feasibility and high quality of chromatin accessibility profiling (using Omni-ATAC) as well as profiling of histones and histone modifications using ChIP-seq, CUT&RUN, and CUT&Tag.
Materials
Reagent1X PBS (Phosphate-buffered saline ) ReagentDynabeads™ Protein G for ImmunoprecipitationThermo FisherCatalog #10003D Reagentanti GFP antibodyRocheCatalog #11814460001
ReagentAnti-Histone H3 antibodyAbcamCatalog #ab1791 ReagentAnti-Histone H3 (tri methyl K4) antibody - ChIP gradeAbcamCatalog #Ab8580 ReagentAnti-Histone H3 (tri methyl K36) antibody - ChIP gradeAbcamCatalog #Ab9050 Reagentllumina Tagment DNA TDE1 Enzyme and Buffer KitsilluminaCatalog #20034197 ReagentIDT® for Illumina® DNA/RNA UD Indexes Set A Tagmentation (96 Indexes 96 Samples)illuminaCatalog #20027213 ReagentCUTANA™ pAG-Tn5 for CUT&TagEpiCypherCatalog #15-1017

Protocol materials
ReagentChIP DNA Clean & ConcentratorZymo ResearchCatalog #D5205
ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
ReagentDynabeads™ Pan Mouse IgGInvitrogen - Thermo FisherCatalog #11041
ReagentDynabeads™ Protein G for ImmunoprecipitationThermo FisherCatalog #10003D
ReagentOvation® Ultralow V2 DNA-Seq Library Preparation KitTecanCatalog # 0344NB-A01
ReagentPureProteome™ Magnetic StandMerck MilliporeSigma (Sigma-Aldrich)Catalog #LSKMAGS08
ReagentTRI ReagentZymo ResearchCatalog #R2050-1-50
ReagentQubit RNA HS Assay KitThermo Fisher ScientificCatalog #Q32852
ReagentDNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
ReagentIDT for Illumina Nextera DNA Unique Dual IndexesIllumina, Inc.Catalog #20027213
ReagentNEBNext High-Fidelity 2X PCR Master Mix - 50 rxnsNew England BiolabsCatalog #M0541S
ReagentCapillary electrophoresis instrument (e.g. Agilent Tapestation 4200)
ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906
ReagentpAG/MNase plasmidaddgeneCatalog #123461
ReagentQuick-DNA Microprep Plus KitZymo ResearchCatalog #D4074
ReagentAgencourt AmPure XP beadsCatalog #A63880
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentProteinase K Solution (20 mg/mL)Thermo Fisher ScientificCatalog #AM2548
ReagentCUTANA™ pAG-Tn5 for CUT&TagEpiCypherCatalog #15-1017
ReagentCUTANA™ High Fidelity 2X PCR Master MixEpiCypherCatalog #15-1018
ReagentAgencourt AmPure XP beadsCatalog #A63880
Reagent100ml TE Buffer [1X], pH 8.0, Low EDTA (Tris-EDTA; 10mM Tris base, 0.1mM EDTA)G-BiosciencesCatalog #786-150
ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
ReagentDynabeads™ Pan Mouse IgGInvitrogen - Thermo FisherCatalog #11041
Reagentanti GFP antibodyRocheCatalog #11814460001
ReagentcOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #4693132001
ReagentNEBNext® RNA Depletion Core Reagent Set with RNA Sample Purification BeadsNew England BiolabsCatalog #E7870L
ReagentNEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®New England BiolabsCatalog #E7760
Buffer preparation
Buffer preparation
Homogenization/Wash Buffer
  • 40 mM HEPES, pH 7.5
  • 120 mM KCl
  • 0.4% NP40 (IGEPAL)

Dilution Buffer [cold]
  • 40 mM HEPES, pH 7.5
  • 120 mM KCl

Bead Washing Buffer [cold]
  • 1X Phosphate Buffer Saline (PBS) buffer, pH 7.4
  • 2.5 mM MgCl2
  • 0.02% Tween-20
Drosophila stocks
Drosophila stocks
Generate flies expressing GFPKASH protein in the cell type of interest by crossing UAS-GFPKASH or QUAS-GFPKASH flies with the appropriate Gal4 or QF driver. Confirm expression patterns by microscopy. We typically generate recombinant flies expressing the driver and GFPKASH where both transgenes are homozygous (two copies) because this is convenient for expanding the flies, and we have found that the IP efficiency improves with higher expression. However, we can also obtain nuclei from flies expressing only a single copy of the driver and GFPKASH, even when combining these with other UAS-transgenes such as RNAi or overexpression.
Bead-antibody coupling
Bead-antibody coupling
30m
30m
Note: The following amounts of Dynabeads and antibody are for 400 fly heads. These amounts can be scaled depending on input.

Incubate 20 μL/reaction of ReagentDynabeads™ Pan Mouse IgGInvitrogen - Thermo FisherCatalog #11041 in 1 mL Bead Washing Buffer for 10 min at RT with constant rotation.
Transfer tube to magnet stand and remove supernatant.

  • For this step, and all steps involving the magnet - briefly spin tubes in a microcentrifuge to remove any liquid from the cap before placing tube on the magnet stand.
  • We use a 1 mL pipettor to remove the supernatant at all bead/magnet steps.
  • Wait at least 1 min to make sure all beads are cleared from solution before pipetting liquid out.

ReagentPureProteome™ Magnetic StandSigma AldrichCatalog #LSKMAGS08
Resuspend beads in 500 μL Bead Washing Buffer and add 2 μg Reagentanti GFP antibodySigma AldrichCatalog #11814460001 .
Incubate at room temperature for 30 min with constant rotation.
Transfer tube to magnet and remove supernatant.
Resuspend beads in 100 μL 0.1% Homogenization/Wash Buffer (mix 3 parts dilution buffer and 1 part homogenization/wash buffer for a final NP-40 concentration of 0.1%).

Note
The resuspension volume can be altered depending on how many samples are being processed. Aim for 50 μL/sample. e.g., if you are processing 4 samples for NIE, resuspend in 200 μL 0.1% homogenization/wash buffer.

Homogenization
Homogenization
10m
10m

Note
Homogenization notes:
  • Always keep homogenizer on ice
  • Use 3 mL of homogenization buffer for any number up to 400 fly heads - from flies snap frozen in liquid nitrogen and stored at -80°C
  • Fresh samples can also be used e.g., partially dissecting tissues from larvae, whole embryos - but nuclei can be isolated successfully from frozen flies and this is our standard approach for neuronal cell types in the adult head

Add 3 mL of Homogenization/Wash Buffer and 100 μL 25X ReagentcOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #4693132001 to a Dounce homogenizer. Keep on ice.
Transfer frozen flies into 15 mL conical tubes. Pre-chill the sieves, flies, and paint brush using liquid nitrogen.
Vortex conical tubes containing flies for 3 seconds, then place tubes back into liquid nitrogen. Repeat 4 more times to separate fly heads from bodies.

Keep sieves cold by pouring a small amount of liquid nitrogen over them between vortexing tubes.
Pour flies into pre-cooled top sieve and tap top sieve so fly heads fall into middle sieve. Remove top sieve and tap middle sieve to get rid of any smaller debris.
Transfer separated fly heads from middle sieve into 7 mL glass Dounce homogenizer using a paint brush chilled using liquid nitrogen.
Grind samples in Dounce homogenizer with 5 "loose" pestle strokes.
  • Homogenize slowly, lifting pestle all the way out to the wide bulb each time
  • Twisting the pestle in the bottom may improve homogenization
Incubate samples on ice for 1 min.
Grind samples with 5 "tight" pestle strokes.
Filter homogenate using 40 μm cell strainer or Miracloth and glass funnel into two microcentrifuge tubes.
Note
If using Miracloth:
  • Cut ends off 1000 μL pipette tips (one per sample) using scissors sterilized with Ethanol - tips are cut so fly heads can be pipetted easily
  • Put small glass funnel into microcentrifuge tube
  • Cut a square of Miracloth (large enough to fit into funnel and extend slightly over the sides) and place into funnel
  • Pipette half of homogenate through the Miracloth and funnel into one microcentrifuge tube using cut tip (head debris will stay in Miracloth, while nuclei will pass through)
  • Transfer funnel and Miracloth into second microcentrifuge tube, and pipette rest of homogenate into tube

Centrifuge homogenate at 800 x g for 5 min at 4°C.
Carefully decant supernatant containing cell debris and resuspend the two pellets from each sample in 0.5 mL 0.1% homogenization/wash buffer total, combining the two pellets into one microcentrifuge tube.
Pipette combined pellets up and down ~10 times, or until pellet is completely dispersed.
Add 20 μL 25X protease inhibitor to each tube.
Nuclei - bead incubation
Nuclei - bead incubation
1h 30m
1h 30m
Add 50 μL antibody-bound beads into each tube that has homogenate.

Or, split antibody-bound beads evenly between all tubes depending on the resuspension volume.
Incubate nuclei and antibody-bound beads for 60 min at 4°C with constant rotation.
Remove supernatant using a magnet.
Gently resuspend bead-bound nuclei with 1 mL 0.1% homogenization/wash buffer.
Incubate for 5 min at 4 °C with constant rotation.

Repeat wash steps 23-24 two more times.

If proceeding to CUT&RUN, use CUT&RUN Dig-wash Buffer on final wash (See Janssens & Henikoff protocol linked below in CUT&RUN section).
After final wash, samples contain bead-bound GFP-expressing nuclei and can be used subsequently for RNA-seq, Omni-ATAC, ChIP-seq, CUT&Tag, or CUT&RUN.
CUT&RUN
CUT&RUN
Our CUT&RUN protocol is based on the following protocol, proceeding from nuclei-bead incubation directly to Step 9: "Permeabilize cells and bind primary antibodies":
CITATION
Derek Janssens, Steven Henikoff. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers. protocols.io.
LINK
https://protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn
We make the following modifications to the linked protocol:

  • We purify our own pAG/MNase enzyme (ReagentpAG/MNase plasmidaddgeneCatalog #123461 ) using the protocol outlined in the following paper:
CITATION
Meers MP, Bryson TD, Henikoff JG, Henikoff S (2019). Improved CUT&RUN chromatin profiling tools..
LINK
https://doi.org/10.7554/eLife.46314
  • Step 11: Use 150 μL antibody buffer.
  • Step 13: Place microcentrifuge tubes containing samples into 50 mL conical tubes. Place conical tubes on a roller mixer overnight. This prevents beads from settling and ensures liquid stays in motion at this small volume.
  • Skip “Bind secondary antibody as required” section.
  • Follow “Chromatin Digestion and Release Option 1: Standard CUT&RUN”
  • Step 32: Place samples on a metal cold block cooled to 0°C in an ice bath (a heater block from a dry bath works for this purpose).
  • Step 34: Incubation time should be assayed for each target using a pilot experiment We have used the following incubation times:
ABC
TargetAntibodyIncubation time (min)
GFPAbcam, ab65565
H3K4me2/3Abcam, ab858020
H3K36me3Abcam, ab905020
H3K9me3Abcam, ab17691620
H3K27me3EpiCypher, 13-005520
H3K4me1Abcam, ab889530
H3K4me2Thermo Fisher, 71079630
H3K4me3EpiCypher, 13-004130
RNA Pol II CTD Millipore Sigma, 0506232
Antibody incubation time table.
  • Step 35: Make master mix of stop buffer ahead of time that contains spike-in DNA for normalization.
  • Step 36: Halfway through 30 min incubation, remove tubes from heat block and tap to resuspend beads, then place back on heat block.
  • Step 58: Increasing original sample volume with water and scaling all steps to the increased volume can reduce sample loss when pipetting aqueous layer (if not using phase-lock tube).

For DNA library preparation, use the following protocol:
CITATION
Nan Liu. Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645). protocols.io.
LINK
https://protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-bagaibse
We make the following modification to the linked protocol:
  • Step 16: Before proceeding with PCR amplification, take 1/5 of the reaction volume for qPCR to determine appropriate cycle number for each sample. Choose a cycle number corresponding to 1/2 - 2/3 up the amplification curve within the exponential phase of the qPCR.
AB
ComponentVolume (μL)
Adaptor Ligated Fragments2.6
NEBNext Ultra II Q5 Master Mix3
Index Primer/i7 Primer0.2
Universal PCR Primer/i5 Primer0.2
20X EvaGreen0.3
1x qPCR reaction mix to determine PCR cycle number.

RNA-seq
RNA-seq
Resuspend beads in ReagentTRI ReagentSigma AldrichCatalog #R2050-1-50 and purify according to the manufacturers' instructions. We typically use Direct-Zol microprep kit, eluting in 15 μL, and quantify 2 μL of eluted RNA using ReagentQubit RNA HS Assay KitSigma AldrichCatalog #Q32852 .
Libraries can be generated using:
  • rRNA depletion kitReagentNEBNext® RNA Depletion Core Reagent Set with RNA Sample Purification BeadsNew England BiolabsCatalog #E7870L
  • RNA library prep kit ReagentNEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®New England BiolabsCatalog #E7760
  • Custom ssDNA probes for rRNA depletion from IDT

This kit allows RNA input: [10ng-1ug]. Our custom ribodepletion has reduced Drosophila melanogaster rRNA reads to less than 10% in the raw data. Probe sequences are available upon request. Additionally, we incorporated a qPCR step using 1/5 of the adaptor-ligated cDNA to optimize the cycle number required for the final amplification, see step 30 for details.


Omni-ATAC
Omni-ATAC
Reagents

Omni-ATAC Tagmentation Mix
  • 25 µL 2X buffer
  • 2.5 µL Tn5
  • 16.5 µL PBS
  • 0.5 µL 1% digitonin
  • 0.5 µL 10% Tween-20
  • 5 µL H2O
Begin the Omni-ATAC protocol at this step using the bead-bound nuclei obtained in Step 22.
After third wash, resuspend nuclei in 500 μL Homogenization/Wash Buffer.
Quantify gDNA from 10% of nuclei suspension or count nuclei using hemocytometer.
Note
We typically determine gDNA using ReagentQuick-DNA Microprep Plus KitSigma AldrichCatalog #D4074 kit, and use this to determine the amount of nuclei suspension to use for Omni-ATAC. For comparisons between samples under different experimental conditions, the same amount of nuclei (DNA) should be used.

Based on quantification, aliquot nuclei according to desired input amount. We have successfully used 50 ng or 100 ng DNA equivalent for Omni-ATAC, but it is likely that much lower input DNA levels will also work well using this protocol.
Using magnet, remove supernatant and resuspend nuclei in 50 μL of Omni-ATAC tagmentation mix.

Perform Omni-ATAC as described in this publication:
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 (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues..
LINK
https://doi.org/10.1038/nmeth.4396

Incubate reaction for 30 minutes at 37C in a thermal shaker using 1000 RPM shaking speed.
Purify DNA usingReagentDNA Clean & Concentrator™-5Sigma AldrichCatalog #D4003 and elute in 15 μL elution buffer (from the Zymo kit).
PCR amplify Omni-ATAC libraries:

25 μL ReagentNEBNext High-Fidelity 2X PCR Master Mix - 50 rxnsSigma AldrichCatalog #M0541S
15 μL purified DNA
10 μL ReagentIDT for Illumina Nextera DNA Unique Dual IndexesSigma AldrichCatalog #20027213
Amplify for 5 cycles.

ABC
TemperatureTime
72°C5 min
98°C30 sec
98°C10 sec5 cycles
63°C30 sec
72°C1 min
Thermocycler conditions.

Place reaction on ice.
Determine additional PCR cycles using qPCR:
AB
ComponentVolume (μL)
25 uM Nextera P1 0.25
25 uM Nextera P20.25
100X Syber Green I 0.09
NEBnext 2X 5
diH2O 4.4
1x qPCR reaction mix.

Purify DNA using ReagentAgencourt AmPure XP beadsSigma AldrichCatalog #A63880 using double size selection (0.5 - 1X ratio).
Assess tagmentation patterns usingReagentCapillary electrophoresis instrument (e.g. Agilent Tapestation 4200)Sigma Aldrich
Libraries can be directly sequenced after this step.
ChIP-seq
ChIP-seq
Reagents

A1 Buffer
  • 15 mM HEPES
  • 15 mM NaCl
  • 60 mM KCl
  • 4 mM MgCl2
  • 0.5% Triton X-100

Nuclei Lysis Buffer
  • 50 mM Tris
  • 10 mM EDTA
  • 1% SDS

X-ChIP Dilution Buffer
  • 16.7 mM Tris-HCl, pH 8.0
  • 167 mM NaCl
  • 1% Triton X-100
  • 1.2 mM EDTA

X-ChIP Elution Buffer
  • 100 mM NaHCO3
  • 1% SDS

Low Salt Buffer
  • 20 mM Tris-HCl, pH 8.0
  • 150 mM NaCl
  • 0.1% SDS
  • 1% Triton X-100
  • 2 mM EDTA

High Salt Buffer
  • 20 mM Tris-HCl, pH 8.0
  • 500 mM NaCl
  • 0.1% SDS
  • 1% Triton X-100
  • 2 mM EDTA

LiCl Wash Buffer
  • 10 mM Tris-HCl, pH 8.0
  • 250 mM LiCl
  • 0.1% Na-Deoxycholate
  • 0.1% NP-40 or IGEPAL
  • 1 mM EDTA

TE Buffer
  • 10 mM Tris-HCl, pH 8.0
  • 1 mM EDTA

Begin the ChIP-seq protocol at this step using the bead-bound nuclei obtained in Step 22.
After third wash, use a magnet to remove supernatant.
Resuspend bead-bound nuclei in 1 mL A1 Buffer.
Add ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeSigma AldrichCatalog #28906 to a final concentration of 1%. We use these small ampules for ChIP experiments and discard ~2 weeks after opening, storing at 4C.
Rotate for 2 min at RT.
Add Glycine to a final concentration of 125 mM for quenching and rotate for 5 min at RT.
Resuspend in 140 μL of Nuclei Lysis Buffer.
Transfer to sonication tube (MicroTube (6x16mm), AFA fiber with Snap-Cap 520045).
Sonicate chromatin with E220 Covaris.
Conditions: 10 min with 2% duty cycle 105W, 200 CPB
Transfer the sonicated lysate to an eppendorf tube using a magnet to discard beads.
Add X-ChIP Dilution Buffer to make up to 1mL final volume.
Centrifuge supernatant 10min at 20,000 x g at 4ºC.
Transfer supernatant [soluble chromatin] to new centrifuge tube on ice.
Take a 5% fraction (for input prep go to step 61.1) and flash-freeze remaining chromatin in liquid nitrogen or continue to step 62.
Fill up to 200 µL with X-ChIP Elution Buffer
Add 2 µL of ReagentRNase A (10 mg/mL)Sigma AldrichCatalog #EN0531 and incubate at 37ºC for 1 hour

Add 2 µL of ReagentProteinase K Solution (20 mg/mL)Sigma AldrichCatalog #AM2548 and incubate at 55C overnight. It is important to do this incubation step at 55ºC (not higher temp).

Purify DNA using ReagentChIP DNA Clean & ConcentratorSigma AldrichCatalog #D5205 and quantify 2 µL using ReagentQubit 1X dsDNA HS Assay KitSigma AldrichCatalog #Q33230

Divide soluble chromatin based on number of antibodies to be used and fill up each tube to 1 mL with X-ChIP Dilution Buffer. We recommend using ~100ng equivalent of DNA (chromatin) per antibody for histone mark antibodies (eg H3K4me3), but lower amounts may be sufficient for bulk histone (eg histone H3). Higher amounts may be required depending on the epitope of interest.
Add 1 μg antibody of interest and incubate at 4ºC with constant rotation overnight.
Day 2

Wash 25 µL of beads with 1 mL of X-ChIP dilution buffer to get rid of the slurry.
Immuno-precipitate the antibody-chromatin complex with 25 µL G agarose beads (Santa Cruz) for 2 hours at 4ºC.
Wash the beads with the following buffers for 5 min at RT with constant rotation: Low Salt Buffer, High Salt Buffer, LiCl Wash Buffer. Use 1 mL of each wash buffer, and remove supernatant using magnet as in other steps.
After LiCl wash, resuspend beads in 1 mL of TE buffer and transfer to new centrifuge tube (1.5 mL tube).
Incubate for 5 minutes at 4ºC.
Using a magnet, remove supernatant and resuspend beads in 200 µL of X-ChIP Elution Buffer.
Extract the DNA from each ChIP sample obtained at step 60 using the same method as described for the input fraction (5%): steps 51.2 to 51.4 (RNAse, Proteinase K, purification).
Use purified DNA for library construction. We use ReagentOvation® Ultralow V2 DNA-Seq Library Preparation KitSigma AldrichCatalog # 0344NB-A01 and have found that 100 pg and 2 ng of DNA yield comparable libraries.

CUT&Tag
CUT&Tag
Reagents

Wash 150 Buffer
  • 20 mM HEPES, pH 7.5
  • 150 mM NaCl
  • 0.5 mM Spermidine
  • 1X Roche cOmpleteTM, Mini, EDTA-free protease inhibitor (1 tablet/10mL Wash150 buffer)
Store at 4°C for up to 1 week

Digitonin150 Buffer
  • Wash buffer + 0.01% Digitonin
Prepare fresh each day and store at 4°C

Antibody150 Buffer
  • Digitonin buffer + 2 mM EDTA
Prepare fresh each day and store at 4°C

Wash300 Buffer
  • 20 mM HEPES, pH 7.5
  • 300 mM NaCl
  • 0.5 mM Spermidine
  • 1X Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor (1 tablet/10 mL Wash300 buffer)
Store at 4°C for up to 1 week

Digitonin300 Buffer
  • Wash 300 Buffer + 0.01% Digitonin
Prepare fresh each day and store at 4°C

Tagmentation Buffer
  • Wash buffer + 10 mM MgCl2
Store at 4°C for up to 1 week

TAPS Buffer
  • 10 mM TAPS, pH 8.5
  • 0.2 mM EDTA
Store at RT for up to 6 months

SDS Release Buffer
  • 10 mM TAPS, pH 8.5
  • 0.1% SDS
Store at RT for up to 6 months

SDS Quench Buffer
  • 0.67% Triton-X 100 in Molecular grade H2O
Store at RT for up 6 months
If nuclei will be used for CUT&Tag, perform the nuclear immuno-enrichment (starting at step 3) using ReagentDynabeads™ Pan Mouse IgGSigma AldrichCatalog #11041 instead of ReagentDynabeads™ Protein G for ImmunoprecipitationSigma AldrichCatalog #10003D since Protein G coupled dynabeads might interfere with downstream steps in CUT&Tag.

After third wash, remove supernatant using a magnet and wash nuclei with 1 mL of cold Antibody 150 Buffer three times.
Using magnet, remove supernatant, resuspend bead-bound nuclei in 50 μL Antibody 150 Buffer and transfer to PCR tube
Add 0.5 μg Primary Antibody and gently pipette up and down to mix.
Incubate for 1 hour at RT at 4C with constant rotation.
Using magnet, remove supernatant, resuspend bead-bound nuclei in 50 µL cold Digitonin 150 Buffer.
Add 0.5 μg secondary antibody.
Incubate for 30 min at RT with constant rotation.
Using a magnet, remove supernatant and add 200 μL cold Digitonin 150 Buffer.
Repeat step 70 two times
Remove from magnet, add 50 μL cold Digitonin 300 Buffer.
Add 2.5 μL ReagentCUTANA™ pAG-Tn5 for CUT&TagSigma AldrichCatalog #15-1017 and pipette up and down to mix.
Incubate samples for 1 hour at RT with constant rotation.
Using a magnet, remove supernatant and add 200 μL cold Digitonin 300 Buffer. Thoroughly resuspend by pipetting, return to magnet then pipet to remove supernatant.
Repeat previous step for total of two washes.
Remove from magnet, add 50 μL cold Tagmentation Buffer.
Incubate for 1 hour at 37°C in thermocycler.
Using a magnet, remove supernatant and resuspend beads in 50 μL RT TAPS Buffer.
Using a magnet, remove supernatant, add 5 μL RT SDS Release Buffer and vortex on max speed for 7 seconds. Quick spin to collect.
Add 15 μL RT SDS Quench Buffer and vortex on max speed.
Add 2 μL each of Universal i5 and barcoded i7 primers (10 μM stocks).
Add 25 μL ReagentCUTANA™ High Fidelity 2X PCR Master MixSigma AldrichCatalog #15-1018 and mix.
Amplify in a thermocycler using the following conditions:
ABC
TemperatureTime
58°C5 min
72°C5 min
98°C45 sec
98°C15 secRepeat 14 - 21 cycles
60°C10 sec
72°C1 min
4°Chold
Thermocycler conditions.

We have found that 20 cycles yield optimal libraries when a H3K4me3 CUT&Tag reaction is started using nuclei corresponding to 100 ng gDNA .
Clean CUT&Tab libraries using 1.3X ReagentAgencourt AmPure XP beadsSigma AldrichCatalog #A63880 .

Elute DNA in 15 μLReagent100ml TE Buffer [1X], pH 8.0, Low EDTA (Tris-EDTA; 10mM Tris base, 0.1mM EDTA)Sigma AldrichCatalog #786-150 and quantify using ReagentQubit 1X dsDNA HS Assay KitSigma AldrichCatalog #Q33230 .

CUT&Tag libraries are ready for sequencing.
Citations
Step 29
Derek Janssens, Steven Henikoff. CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers
https://protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn
Step 29
Meers MP, Bryson TD, Henikoff JG, Henikoff S. Improved CUT&RUN chromatin profiling tools.
https://doi.org/10.7554/eLife.46314
Step 30
Nan Liu. Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645)
https://protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-bagaibse
Step 39
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.
https://doi.org/10.1038/nmeth.4396