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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Recipes]
Homogenization/wash [WB] buffer40 mM HEPES, pH 7.5120 mM KCl0.4% NP40 (IGEPAL)Dilution buffer [cold]40 mM HEPES, pH 7.5120 mM KClBead washing buffer [cold]1X Phosphate Buffer Saline (PBS) buffer, pH 7.42.5 mM MgCl2## Omni-ATAC Omni-ATAC tagmentation mix25 µL 2X buffer2.5 µL Tn516.5 µL PBS0.5 µL 1% digitonin0.5 µL 10% Tween-205 µL H2O## ChIP-seqA1 buffer15 mM HEPES15 mM NaCl60 mM KCl4 mM MgCl20.5% Triton X-100Nuclei Lysis Buffer50 mM Tris10 mM EDTA1% SDSX-ChIP dilution buffer16.7 mM Tris-HCl, pH 8.0167 mM NaCl1% Triton X-1001.2 mM EDTAX-ChIP elution buffer100 mM NaHCO31% SDSLow Salt Buffer20 mM Tris-HCl, pH 8.0150 mM NaCl0.1% SDS1% Triton X-1002 mM EDTAHigh Salt Buffer20 mM Tris-HCl, pH 8.0500 mM NaCl0.1% SDS1% Triton X-1002 mM EDTALiCl wash buffer10 mM Tris-HCl, pH 8.0250 mM LiCl0.1% Na-Deoxycholate0.1% NP-40 or IGEPAL1 mM EDTATE buffer10 mM Tris-HCl, pH 8.01 mM EDTA## CUT&TagWash 150 buffer20 mM HEPES, pH 7.5150 mM NaCl0.5 mM Spermidine1X Roche cOmpleteTM, Mini, EDTA-free protease inhibitor (1 tablet/10mL Wash150 buffer)Store at 4C for up to 1 weekDigitonin150 bufferWash buffer + 0.01% DigitoninPrepare fresh each day and store at 4CAntibody150 bufferDigitonin buffer + 2 mM EDTAPrepare fresh each day and store at 4CWash300 buffer20 mM HEPES, pH 7.5300 mM NaCl0.5 mM Spermidine1X Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor (1 tablet/10 mL Wash300 buffer)Store at 4C for up to 1 weekDigitonin300 bufferWash 300 Buffer + 0.01% DigitoninPrepare fresh each day and store at 4CTagmentation bufferWash buffer + 10 mM MgCl2Store at 4C for up to 1 weekTAPS buffer10 mM TAPS, pH 8.50.2 mM EDTAStore at RT for up to 6 monthsSDS Release Buffer10 mM TAPS, pH 8.50.1% SDSStore at RT for up to 6 monthsSDS Quench Buffer0.67% Triton-X 100 in Molecular grade H2OStore at RT for up 6 monthsPrimers:Nextera P1: AATGATACGGCGACCACCGAGANextera P2: CAAGCAGAAGACGGCATACGAUniversal i5: AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGIndexed i7-1: CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTIndexed i7-2: CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTIndexed i7-3: CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTIndexed i7-4: CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT
?. [Bead-antibody coupling]
Incubate 40 μL of  in 1 mL Bead washing buffer for 10 min at RT with constant rotation
?. [Bead-antibody coupling]
Resuspend beads in 1 mL Bead washing buffer and add 4 μg
?. [Nuclei - bead incubation]
Split 4 mL lysate into four microcentrifuge (1.5 mL) tubes
?. [Homogenization]
(Always keep homogenizer on ice)(1 mL of homogenization buffer is good for 400 fly heads - from flies snap frozen in liquid nitrogen and stored at -80C)(fresh samples can also be used i.e. 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)Transfer fly samples to 1 mL dounce homogenizer that contains 1 volume of cold homogenization/wash buffer
?. [RNA-seq]
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
?. [Omni-ATAC]
After third wash, resuspend nuclei in 500 μL homogenization/wash buffer
?. [ChIP-seq]
Resuspend bead-bound nuclei in 1 mL A1 buffer
?. [Bead-antibody coupling]
Transfer tube to magnet and remove supernatant. For this step, and all steps involving the magnet - invert the tube several times on the magnet to ensure all beads are bound and remove any remaining supernatant from inside the lid of the tube using a pipettor. We use a 1 mL pipettor to remove the supernatant at all bead/magnet steps.
?. [Bead-antibody coupling]
Resuspend beads in 200 μL 0.1% homogenization/wash buffer (mix 3 parts dilution buffer and 1 part homogenization buffer/wash - final NP-40 concentration of 0.1%)
?. [Bead-antibody coupling]
Transfer tube to magnet and remove supernatant
?. [Homogenization]
Grind samples with 5 "loose" pestle strokes
?. [Homogenization]
Incubate samples on ice for 5 min
?. [Homogenization]
Repeat 5 "loose" pestle strokes and follow with 10 "tight" pestle strokes
?. [Homogenization]
Filter homogenate using a  and add 3 volumes of dilution buffer
?. [Nuclei - bead incubation]
Add a 1/4 of resuspended bead-antibody complex into each tube
?. [Nuclei - bead incubation]
Incubate nuclei-bead-antibody solution for 30 min at 4C with constant rotation
?. [Nuclei - bead incubation]
Using a magnet, remove supernatant
?. [Nuclei - bead incubation]
Gently resuspend bead-bound nuclei with 1 mL homogenization/wash buffer
?. [Nuclei - bead incubation]
Incubate for 5 min at 4C with constant rotation
?. [Nuclei - bead incubation]
Using a magnet, remove supernatant
?. [Nuclei - bead incubation]
Gently resuspend bead-bound nuclei with 1 mL homogenization/wash buffer and transfer to new centrifuge tube
?. [Nuclei - bead incubation]
Incubate for 5 min at 4C with constant rotation
?. [Nuclei - bead incubation]
Repeat wash 1 more time (no need to switch tubes again). At this step, samples contain bead-bound nuclei and can be used for RNA-seq (see step 22), Omni-ATAC (see step 25), ChIP-seq (see step 39), or CUT&Tag (see step 63: note that different magnetic beads should be used for CUT&Tag in step 2).
?. [ChIP-seq]
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.
?. [Omni-ATAC]
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.
?. [Omni-ATAC]
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.
?. [Omni-ATAC]
Using magnet, remove supernatant and resuspend nuclei in 50 μL of Omni-ATAC tagmentation mix
?. [Omni-ATAC]
Perform Omni-ATAC as described in this publication: (Corces, 2017)
?. [ChIP-seq]
Add paraformaldehyde for a final concentration of 1%
?. [CUT&Tag]
Begin the CUT&Tag protocol at this step using the bead-bound nuclei obtained in Step 22.If nuclei will be used for CUT&Tag, use   instead of at STEP 2. Otherwise, the Protein G might interfere with downstream CUT&Tag reagents.CUT&Tag reagents were purchased from Epicypher and the following protocol provided by the company https://www.epicypher.com/content/documents/protocols/cutana-cut&tag-protocol.pdf was slighly modified, starting at Section III
?. [Omni-ATAC]
Incubate reaction for 30 minutes at 37C in a thermal shaker using 1000 RPM shaking speed.
?. [Omni-ATAC]
Purify DNA using and elute in 15 μL elution buffer (from the Zymo kit).
?. [Omni-ATAC]
PCR amplify Omni-ATAC libraries:25 μL 15 μL purified DNA10 μL
?. [Omni-ATAC]
Determine additional PCR cycles using qPCR:qPCR mix                     1 rxn25 uM Nextera P1      0.25 μL25 uM Nextera P2      0.25 μL100X Syber Green I    0.09 μLNEBnext 2X                 5 μLdiH2O                           4.4 μL
?. [Omni-ATAC]
Amplify for 5 cycles 72C 5min98C 30 secThen, 5 cycles of:98C 10 sec63C 30 sec72C 1 min
?. [Omni-ATAC]
Place reaction on ice
?. [Omni-ATAC]
Purify DNA using   using double size selection (0.5-1X ratio)
?. [ChIP-seq]
Fix for 2 minutes at RT with constant rotation
?. [ChIP-seq]
Quench fixing reaction by adding Glycine for a final concentration of 125 mM and rotate for 5 minutes at RT with constant rotation
?. [ChIP-seq]
Using a magnet remove supernatant and resuspend nuclei in 140 μL Nuclei Lysis Buffer
?. [ChIP-seq]
Transfer to Covaris sonication tube
?. [ChIP-seq]
Sonicate chromatin with E220 Covaris, conditions: 10 min, 2% duty cycle, 105W, 200 C.P.B.
?. [ChIP-seq]
Using a magnet to remove the beads, transfer supernatant [soluble chromatin] to new centrifuge tube
?. [ChIP-seq]
Centrifuge sample for 10 min at 4C with 14000xG
?. [ChIP-seq]
Transfer supernatant [soluble chromatin] to new tube
?. [ChIP-seq]
Take 5% fraction for input (see 48.1 for input DNA prep) and flash-freeze remaining soluble chromatin (or continue to 48)
?. [ChIP-seq]
Fill tube up to 1 mL using XChIP- dilution buffer
?. [ChIP-seq]
If flash-frozen, thaw soluble chromatin
?. [ChIP-seq]
For 50 μL of input, add 150 μL of X-ChIP elution buffer
?. [ChIP-seq]
Add 1 μL of   per 100 μL eluted sample and incubate at 37C for 1 hour
?. [ChIP-seq]
Add 1 μL of   per 100 μL eluted sample and incubate at 55C overnight
?. [ChIP-seq]
Purify DNA with   and quantify DNA using
?. [ChIP-seq]
Split soluble chromatin according to number of antibodies used
?. [ChIP-seq]
Fill up to 1 mL with X-ChIP dilution buffer
?. [ChIP-seq]
Add antibody of interest and incubate at 4C overnight with constant rotation
?. [ChIP-seq]
Day 2Wash 25 μL with 1 mL of X-ChIP dilution buffer for 5 min at RT with constant rotation
?. [ChIP-seq]
Using magnet, remove supernatant and resuspend beads in 25 μL X-ChIP dilution buffer
?. [ChIP-seq]
Add resuspended beads to solution with soluble chromatin and antibody and incubate for 2 hours at 4C with constant rotation
?. [ChIP-seq]
Using a magnet, wash beads for 5 min at 4C with constant rotations using the following buffers:Low Salt BufferHigh Salt BufferLiCl Wash buffer
?. [ChIP-seq]
After the third wash, resuspend beads in 1 mL TE buffer and transfer to new centrifuge tube
?. [ChIP-seq]
Incubate sample for 5 min at 4C with constant rotation
?. [ChIP-seq]
Using a magnet, remove supernatant and resuspend with 200 μL X-ChIP elution buffer
?. [ChIP-seq]
Repeat purification steps as input prep sample (step 47.2)
?. [ChIP-seq]
Construct ChIP-seq libraries using the same amount of DNA for each library following manufacturer's instructions.
?. [CUT&Tag]
After third wash, resuspend bead-bound nuclei in 1 mL of Antibody150 buffer
?. [CUT&Tag]
Using magnet, remove supernatant and resuspend bead-bound nuclei in 50 μL of Antibody 150 buffer
?. [CUT&Tag]
Transfer sample to PCR tube
?. [CUT&Tag]
Add 0.5 ug Primary antibody and resuspend
?. [CUT&Tag]
Incubate tubes on nutator overnight at 4C or for 1 hour at RT
?. [CUT&Tag]
Using a magnet, remove supernatant and resuspend beads in 50 μL cold Digitonin 150 buffer
?. [CUT&Tag]
Incubate for 30 min at RT with constant rotation
?. [CUT&Tag]
Using a magnet, remove supernatant and add 200 μL cold Digitonin 150 buffer
?. [CUT&Tag]
Add 0.5 ug secondary antibody (matched to primary antibody animal source)
?. [CUT&Tag]
While samples are on the magnet, remove the supernatant and repeat step 70 two more times
?. [CUT&Tag]
Remove tubes from magnet and add 50 μL cold Digitonin 300 buffer
?. [CUT&Tag]
Add 2.5 μL  and resuspend by gently pipetting up and down
?. [CUT&Tag]
Incubate samples for 30 min at RT with constant rotation
?. [CUT&Tag]
Using magnet, remove supernatant and add 200 μL Tagmentation buffer
?. [CUT&Tag]
Incubate samples for 1 hour at 37C in a thermocycler
?. [CUT&Tag]
Using magnet, remove supernatant and resuspend beads in 50 μL RT TAPS buffer
?. [CUT&Tag]
Using a magnet, remove supernatant and add 5 μL RT SDS Release Buffer
?. [CUT&Tag]
Vortex on max speed for 7 seconds
?. [CUT&Tag]
Incubate samples for 1 hour at 58C in a thermocycler
?. [CUT&Tag]
Add 15 μL RT SDS Quench Buffer, and vortex on max speed
?. [CUT&Tag]
To each sample, add 2 μL of each universal i5 and barcoded i7 primers (10 uM stocks) and 25 μL
?. [CUT&Tag]
Amplify using the following parameters:a. 58C - 5 minb. 72C - 5 minc. 98C - 45 secd. 98C - 15 sece. 60C - 10 secRepeat d-e for a total of 14-21 cycles When performing H3K4me3 CUT&Tag using 100 ng as starting material, we found 20 cycles to yield optimal libraries.
?. [CUT&Tag]
Clean CUT&Tag libraries using  with a 1.3X ratio and quantify using
?. [Omni-ATAC]
Assess  tagmentation patterns using Libraries can be directly sequenced after this step.
?. [RNA-seq]
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.
?. [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, overexpression.
?. [Omni-ATAC]
Begin the Omni-ATAC protocol at this step using the bead-bound nuclei obtained in Step 22.
?. [ChIP-seq]
Add  to a final concentration of 1%. We use these small ampules for ChIP experiments and discard ~2 weeks after opening, storing at 4C.
?. [ChIP-seq]
Rotate for 2 min at RT
?. [ChIP-seq]
Add Glycine to a final concentration of 125 mM for quenching and rotate for 5 min at RT
?. [ChIP-seq]
Resuspend in 140 uL of Nuclei Lysis Buffer
?. [ChIP-seq]
Transfer to sonication tube (MicroTube (6x16mm), AFA fiber with Snap-Cap 520045)
?. [ChIP-seq]
Sonicate chromatin with E220 CovarisConditions: 10 min with 2% duty cycle 105W, 200 CPB
?. [ChIP-seq]
Transfer the sonicated lysate to an eppendorf tube using a magnet to discard beads
?. [ChIP-seq]
Add  X-ChIP dilution buffer to make up to 1mL final volume.
?. [ChIP-seq]
Centrifuge supernatant 10min at 20,000 x g at 4C.
?. [ChIP-seq]
Transfer supernatant [soluble chromatin] to new centrifuge tube on ice
?. [ChIP-seq]
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
?. [ChIP-seq]
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.
?. [ChIP-seq]
Add 1 μg antibody of interest and incubate at 4C with constant rotation overnight
?. [ChIP-seq]
Day 2
?. [ChIP-seq]
Fill up to 200 µL with X-ChIP elution buffer
?. [ChIP-seq]
Add 2 µL of  and incubate at 37C for 1 hour
?. [ChIP-seq]
Add 2 µL of  and incubate at 55C overnight. It is important to do this incubation step at 55C (not higher temp).
?. [ChIP-seq]
Purify DNA using  and quantify 2 µL using
?. [ChIP-seq]
Wash 25  µL of beads with 1 mL of X-ChIP dilution buffer to get rid of the slurry
?. [ChIP-seq]
Immuno-precipitate the antibody-chromatin complex with 25  µL G agarose beads (Santa Cruz) for 2 hours at 4C
?. [ChIP-seq]
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.
?. [ChIP-seq]
After LiCl wash, resuspend beads in 1 mL of TE buffer and transfer to new centrifuge tube (1.5 mL tube).
?. [ChIP-seq]
Incubate for 5 minutes at 4C
?. [ChIP-seq]
Using a magnet, remove supernatant and resuspend beads in 200 µL of X-ChIP Elution buffer
?. [ChIP-seq]
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).
?. [CUT&Tag]
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.
?. [CUT&Tag]
After third wash, remove supernatant using a magnet and wash nuclei with 1 mL of cold Antibody 150 buffer three times
?. [CUT&Tag]
Using magnet, remove supernatant, resuspend bead-bound nuclei in 50 μL Antibody 150 buffer and transfer to PCR tube
?. [CUT&Tag]
Add 0.5 μg Primary antibody and gently pipette up and down to mix
?. [CUT&Tag]
Incubate for 1 hour at RT at 4C with constant rotation
?. [CUT&Tag]
Using magnet, remove supernatant,  resuspend bead-bound nuclei in 50  µL cold Digitonin 150 buffer
?. [CUT&Tag]
Add 0.5 μg Secondary antibody
?. [CUT&Tag]
Incubate for 30 min at RT with constant rotation
?. [CUT&Tag]
Using a magnet, remove supernatant and add 200 μL cold Digitonin 150 Buffer
?. [CUT&Tag]
Repeat step 70 two times
?. [CUT&Tag]
Remove from magnet, add 50 μL cold Digitonin 300 buffer
?. [CUT&Tag]
Add 2.5 μL  and pipette up and down to mix
?. [CUT&Tag]
Incubate samples for 1 hour at RT with constant rotation
?. [CUT&Tag]
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
?. [CUT&Tag]
Repeat previous step for total of two washes
?. [CUT&Tag]
Remove from magnet, add 50 μL cold Tagmentation Buffer
?. [CUT&Tag]
Incubate for 1 hour at 37C in thermocycler
?. [CUT&Tag]
Using a magnet, remove supernatant and resuspend beads in 50 μL RT TAPS Buffer
?. [CUT&Tag]
Using a magnet, remove supernatant, add 5 uL RT SDS Release Buffer and vortex on max speed for 7 seconds. Quick spin to collect
?. [CUT&Tag]
Add 15 μL RT SDS Quench Buffer and vortex on max speed.
?. [CUT&Tag]
Add 2 μL each of Universal i5 and barcoded i7 primers (10 μM stocks)
?. [CUT&Tag]
Add 25 μL   and mix
?. [CUT&Tag]
Amplify in a thermocycler using the following conditons:a. 58C - 5 minb. 72C - 5 minc. 98C - 45 secd. 98C - 15 sece. 60C - 10 secf. Repeat d-e for a total of 14-21.g. 72C - 1minh. hold at 4CWe have found that 20 cycles yield optimal libraries when a H3K4me3 CUT&Tag reaction is started using nuclei corresponding to 100 ng gDNA
?. [CUT&Tag]
Clean CUT&Tab libraries using 1.3X am
?. [CUT&Tag]
Elute DNA in 15 μL and quantify using
?. [ChIP-seq]
Use purified DNA for library construction. We use  and have found that 100 pg and 2 ng of DNA yield comparable libraries.
?. [CUT&Tag]
CUT&Tag libraries are ready for sequencing