TITLE: In vivo tissue-specific chromatin profiling in Drosophila
AUTHORS: Vikki M. Weake, Juan P P Jauregui-Lozano, Sarah E McGovern

[DESCRIPTION]
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 in the heart of many human diseases, which most of them 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 as well as profiling of histones and histone modifications, using Omni-ATAC and ChIP-seq, respectively. Last, we describe a modification to the nuclei purification protocol that allows for application of recently developed CUT&Tag and demonstrate that CUT&Tag outperforms traditional ChIP-seq, although protocol might require further optimization.

[GUIDELINES]
References:
Corces, M., Trevino, A., Hamilton, E.et al.An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.Nat Methods14,959–962 (2017). https://doi.org/10.1038/nmeth.4396

https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-cut-and-tag-assays

[STEPS]
SECTION: Recipes
1.  
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

## Omni-ATAC
 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

## ChIP-seq
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

## CUT&Tag
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 4C for up to 1 week

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

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

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 4C for up to 1 week

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

Tagmentation buffer
Wash buffer + 10 mM MgCl2
Store at 4C 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



Primers:
Nextera P1: AATGATACGGCGACCACCGAGA
Nextera P2: CAAGCAGAAGACGGCATACGA
Universal i5: AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG
Indexed i7-1: CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT
Indexed i7-2: CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGT
Indexed i7-3: CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT
Indexed i7-4: CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT




SECTION: Bead-antibody coupling
3. 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     in 1 mL bead washing buffer for 10 min at RT with constant rotation.

SECTION: Bead-antibody coupling
5. Resuspend beads in 1 mL bead washing buffer and add 2 μg   .

SECTION: Nuclei - bead incubation
22. 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.
SECTION: Homogenization
9. 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   to a Dounce homogenizer. Keep on ice.

SECTION: RNA-seq
29. Resuspend beads in   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   

SECTION: Omni-ATAC
32. After third wash, resuspend nuclei in 500 μL homogenization/wash buffer
SECTION: ChIP-seq
46. Resuspend bead-bound nuclei in 1 mL A1 buffer
SECTION: Bead-antibody coupling
4. 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.

  

SECTION: Bead-antibody coupling
8. 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%).

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.
SECTION: Bead-antibody coupling
7. Transfer tube to magnet and remove supernatant.
SECTION: Homogenization
14. 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
SECTION: Homogenization
15. Incubate samples on ice for 1 min.
SECTION: Homogenization
16. Grind samples with 5 "tight" pestle strokes.
SECTION: Homogenization
17. Filter homogenate using 40 μm cell strainer or Miracloth and glass funnel into two microcentrifuge tubes.

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
SECTION: Nuclei - bead incubation
23. Incubate nuclei and antibody-bound beads for 60 min at 4°C with constant rotation.
SECTION: Nuclei - bead incubation
24. Remove supernatant using a magnet.
SECTION: Nuclei - bead incubation
25. Gently resuspend bead-bound nuclei with 1 mL 0.1% homogenization/wash buffer.
SECTION: Nuclei - bead incubation
26. Incubate for 5 min at 4 °C with constant rotation.

SECTION: Nuclei - bead incubation
27. Repeat wash steps 23-24 two more times.
SECTION: Nuclei - bead incubation
28. 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.
SECTION: ChIP-seq
45. 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.
SECTION: Omni-ATAC
33. Quantify gDNA from 10% of nuclei suspension or count nuclei using hemocytometer. We typically determine gDNA using    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.

SECTION: Omni-ATAC
34. 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.
SECTION: Omni-ATAC
35. Using magnet, remove supernatant and resuspend nuclei in 50 μL of Omni-ATAC tagmentation mix


SECTION: Omni-ATAC
36. Perform Omni-ATAC as described in this publication: (Corces, 2017)
SECTION: Omni-ATAC
37. Incubate reaction for 30 minutes at 37C in a thermal shaker using 1000 RPM shaking speed.
SECTION: Omni-ATAC
38. Purify DNA using  and elute in 15 μL elution buffer (from the Zymo kit).

SECTION: Omni-ATAC
39. PCR amplify Omni-ATAC libraries:

25 μL   
15 μL purified DNA
10 μL    
 
SECTION: Omni-ATAC
42. Determine additional PCR cycles using qPCR:
qPCR mix                     1 rxn
25 uM Nextera P1      0.25 μL
25 uM Nextera P2      0.25 μL
100X Syber Green I    0.09 μL
NEBnext 2X                 5 μL
diH2O                           4.4 μL

SECTION: Omni-ATAC
40. Amplify for 5 cycles 
72C 5min
98C 30 sec
Then, 5 cycles of:
98C 10 sec
63C 30 sec
72C 1 min
SECTION: Omni-ATAC
41. Place reaction on ice
SECTION: Omni-ATAC
43. Purify DNA using    using double size selection (0.5-1X ratio)

SECTION: Omni-ATAC
44. Assess  tagmentation patterns using  Libraries can be directly sequenced after this step. 

SECTION: RNA-seq
30. Libraries for RNA-seq can be generated using . This kit allows RNA inputs : [10 pg- 10 ng]. This library kit has ribodepletion step incorporated into the protocol using Drosophila anyDeplete, and libraries are therefore total nuclear RNA depleted for rRNA (not mRNA). We also recommend to use a library kit that has an in-solution DNAse step as part of the initial protocol because in our hands, the gDNA removal in the Direct-Zol kit is not 100% efficient.

SECTION: Drosophila stocks
2. 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. 
SECTION: Omni-ATAC
31. Begin the Omni-ATAC protocol at this step using the bead-bound nuclei obtained in Step 22.
SECTION: ChIP-seq
47. Add   to a final concentration of 1%. We use these small ampules for ChIP experiments and discard ~2 weeks after opening, storing at 4C.

SECTION: ChIP-seq
48. Rotate for 2 min at RT
SECTION: ChIP-seq
49. Add Glycine to a final concentration of 125 mM for quenching and rotate for 5 min at RT
SECTION: ChIP-seq
50. Resuspend in 140 uL of Nuclei Lysis Buffer 
SECTION: ChIP-seq
51. Transfer to sonication tube (MicroTube (6x16mm), AFA fiber with Snap-Cap 520045)
SECTION: ChIP-seq
52. Sonicate chromatin with E220 Covaris
Conditions: 10 min with 2% duty cycle 105W, 200 CPB
SECTION: ChIP-seq
53. Transfer the sonicated lysate to an eppendorf tube using a magnet to discard beads
SECTION: ChIP-seq
54. Add  X-ChIP dilution buffer to make up to 1mL final volume.
SECTION: ChIP-seq
55. Centrifuge supernatant 10min at 20,000 x g at 4C.
SECTION: ChIP-seq
56. Transfer supernatant [soluble chromatin] to new centrifuge tube on ice
SECTION: ChIP-seq
57. Take a 5% fraction (for input prep go to step 51.1) and flash-freeze remaining chromatin in liquid nitrogen or continue to step 52
SECTION: ChIP-seq
58. 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.
SECTION: ChIP-seq
59. Add 1 μg antibody of interest and incubate at 4C with constant rotation overnight
SECTION: ChIP-seq
60. Day 2

SECTION: ChIP-seq
57.1. Fill up to 200 µL with X-ChIP elution buffer
SECTION: ChIP-seq
57.2. Add 2 µL of   and incubate at 37C for 1 hour

SECTION: ChIP-seq
57.3. Add 2 µL of   and incubate at 55C overnight. It is important to do this incubation step at 55C (not higher temp).

SECTION: ChIP-seq
57.4. Purify DNA using   and quantify 2 µL using   

SECTION: ChIP-seq
61. Wash 25  µL of beads with 1 mL of X-ChIP dilution buffer to get rid of the slurry
SECTION: ChIP-seq
62. Immuno-precipitate the antibody-chromatin complex with 25  µL G agarose beads (Santa Cruz) for 2 hours at 4C
SECTION: ChIP-seq
63. 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.
SECTION: ChIP-seq
64. After LiCl wash, resuspend beads in 1 mL of TE buffer and transfer to new centrifuge tube (1.5 mL tube).
SECTION: ChIP-seq
65. Incubate for 5 minutes at 4C
SECTION: ChIP-seq
66. Using a magnet, remove supernatant and resuspend beads in 200 µL of X-ChIP Elution buffer
SECTION: ChIP-seq
67. 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, proteinaseK, purification).
SECTION: CUT&amp;Tag
69. If nuclei will be used for CUT&Tag, perform the nuclear immuno-enrichment (starting at step 3) using   instead of  since Protein G coupled dynabeads might interfere with downstream steps in CUT&Tag.

SECTION: CUT&amp;Tag
70. After third wash, remove supernatant using a magnet and wash nuclei with 1 mL of cold Antibody 150 buffer three times
SECTION: CUT&amp;Tag
71. Using magnet, remove supernatant, resuspend bead-bound nuclei in 50 μL Antibody 150 buffer and transfer to PCR tube
SECTION: CUT&amp;Tag
72. Add 0.5 μg Primary antibody and gently pipette up and down to mix
SECTION: CUT&amp;Tag
73. Incubate for 1 hour at RT at 4C with constant rotation
SECTION: CUT&amp;Tag
74. Using magnet, remove supernatant,  resuspend bead-bound nuclei in 50  µL cold Digitonin 150 buffer
SECTION: CUT&amp;Tag
75. Add 0.5 μg Secondary antibody
SECTION: CUT&amp;Tag
76. Incubate for 30 min at RT with constant rotation
SECTION: CUT&amp;Tag
77. Using a magnet, remove supernatant and add 200 μL cold Digitonin 150 Buffer
SECTION: CUT&amp;Tag
78. Repeat step 70 two times
SECTION: CUT&amp;Tag
79. Remove from magnet, add 50 μL cold Digitonin 300 buffer 
SECTION: CUT&amp;Tag
80. Add 2.5 μL   and pipette up and down to mix

SECTION: CUT&amp;Tag
81. Incubate samples for 1 hour at RT with constant rotation
SECTION: CUT&amp;Tag
82. Using a magnet, remove supernatant and add 200 μL cold Digitonin 300 buffer. Thoroughly resuspend by pipetting, return to magnet then pipet to remove supe
SECTION: CUT&amp;Tag
83. Repeat previous step for total of two washes
SECTION: CUT&amp;Tag
84. Remove from magnet, add 50 μL cold Tagmentation Buffer 
SECTION: CUT&amp;Tag
85. Incubate for 1 hour at 37C in thermocycler
SECTION: CUT&amp;Tag
86. Using a magnet, remove supernatant and resuspend beads in 50 μL RT TAPS Buffer
SECTION: CUT&amp;Tag
87. Using a magnet, remove supernatant, add 5 uL RT SDS Release Buffer and vortex on max speed for 7 seconds. Quick spin to collect
SECTION: CUT&amp;Tag
88. Add 15 μL RT SDS Quench Buffer and vortex on max speed. 
SECTION: CUT&amp;Tag
89. Add 2 μL each of Universal i5 and barcoded i7 primers (10 μM stocks)
SECTION: CUT&amp;Tag
90. Add 25 μL    and mix

SECTION: CUT&amp;Tag
91. Amplify in a thermocycler using the following conditons:
a. 58C - 5 min
b. 72C - 5 min
c. 98C - 45 sec
d. 98C - 15 sec
e. 60C - 10 sec
f. Repeat d-e for a total of 14-21.
g. 72C - 1min
h. hold at 4C

We have found that 20 cycles yield optimal libraries when a H3K4me3 CUT&Tag reaction is started using nuclei corresponding to 100 ng gDNA 
SECTION: CUT&amp;Tag
92. Clean CUT&Tab libraries using 1.3X am  

SECTION: CUT&amp;Tag
93. Elute DNA in 15 μL  and quantify using   

SECTION: ChIP-seq
68. Use purified DNA for library construction. We use   and have found that 100 pg and 2 ng of DNA yield comparable libraries. 

SECTION: CUT&amp;Tag
94. CUT&Tag libraries are ready for sequencing
SECTION: Homogenization
10. Transfer frozen flies into 15 mL conical tubes. Pre-chill the sieves, flies, and paint brush using liquid nitrogen. 
SECTION: Homogenization
11. 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.
SECTION: Homogenization
12. 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.
SECTION: Homogenization
13. Transfer separated fly heads from middle sieve into 7 mL glass Dounce homogenizer using a paint brush chilled using liquid nitrogen.
SECTION: Homogenization
18. Centrifuge homogenate at 800 x g for 5 min at 4°C.
SECTION: Homogenization
19. 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.
SECTION: Homogenization
21. Add 20 μL 25X protease inhibitor to each tube.
SECTION: Bead-antibody coupling
6. Incubate at room temperature for 30 min with constant rotation.
SECTION: Homogenization
20. Pipette combined pellets up and down ~10 times, or until pellet is completely dispersed.