TITLE: CUT&Tag with Drosophila tissues
AUTHORS: Kami Ahmad, Steven Henikoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a modification of the Benchtop CUT&Tag method (</span><a href="https://dx.doi.org/10.17504/protocols.io.bcuhiwt6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.bcuhiwt6</span></a><span>) for epigenomic profiling of histone modifications and chromatin proteins in dissected Drosophila tissues. Intact unfixed tissues are permeabilized and incubated under conditions where a factor-specific antibody can infiltrate and bind a chromatin protein </span><span style = "font-style:italic;">in situ</span><span>. This antibody is then decorated with a secondary antibody, and this is used to tether a protein AG-Tn5 fusion protein loaded with sequencing adapters. Upon transposome activation by adding Mg2+ ions, DNA is tagmented around the chromatin binding site. Here, dissected imaginal discs are coated with magnetic beads for handling, and about 5 imaginal discs provides high-quality data for histone modifications or for chromatin factor for which high-quality antibodies are available. The protocol is scaled for 8 samples. Performing CUT&Tag takes 2 days from larvae to sequencing-ready libraries.</span></div></div>

[STEPS]
?. [Prepare solutions and beads]
Prepare Wash+ Buffer (50 mL):Add 50 mL of Wash $ buffer solution to a 50 mL conical tube Add 1 large Roche cOmplete EDTA-free tabletsAdd 12.5 µL 2 M Spermidine             final 0.5 mMKeep on ice or store overnight at 4˚C.
?. [Dissect larvae]
Work with a dissecting microscope with tangential illumination. Collect healthy 3rd instar larvae in a glass dish with PBT. Wash off any yeast and food.
?. [Dissect larvae]
Aliquot 200 µL Wash+ buffer into 3 wells of a dissection plate for each genotype.Transfer larvae from PBT to the first well of Wash+ buffer and dissect out imaginal discs. Aim for 4 wing discs or 6 eye discs/sample, so 32 wing discs (or 48 eye discs) for 8 parallel samples. As you dissect, transfer parts to the next wells with Wash+ buffer to minimize contamination with other tissues.
?. [Primary antibody binding]
Add 20 µL Wash+ buffer to each tube of a 8-tube strip. Transfer imaginal discs with forceps to each tube. Add 20 µL ConA bead suspension to each tube and pipette gently to mix. Incubate 10' RT.All successive buffer changes are by grabbing beads with the magnet, pipetting off liquid, and replacing with new buffers. Be careful not to withdraw any beads during washes. Beads are not resuspended or agitated at any time in the procedure to minimize sample loss.
?. [Primary antibody binding]
Place strip on low-volume side of magnet and let bind for 1’. Pipette off the buffer, and replace with 20 µL antibody dilution. Do not resuspend the beads. Remove the strip from the magnet and incubate with no movement 4˚C O/N.
?. [Secondary antibody binding]
Prepare 2˚ antibody dilution in dbe+ buffer. You’ll need 20 µL for each sample at 1:100 dilution, so for 8 reactions with a rabbit 1˚ antibody, aliquot 100 µL dbe+ buffer, add 1 µL 2˚ antibody, and pipette to mix.
?. [Secondary antibody binding]
Place sample strip on low-volume side of magnet and let bind for 1’. Pipette off the 1˚ antibody solution buffer and replace with 20 µL 2˚ antibody dilution. Do not resuspend the beads. Remove the strip from the magnet and incubate with no movement 1 hr at RT.
?. [pAG-Tn5 tethering]
Batches of pAG-Tn5 are calibrated after production. Use the appropriate calibrated dilution for your batch, or recommended dilutions for commercial enzymes.Prepare pAG-Tn5 dilution: For 8 samples, Aliquot 200 µL 300Wash+ buffer, add 1 µL pAG-Tn5 and mix by pipetting. Keep on ice or store at 4˚C.
?. [pAG-Tn5 tethering]
Place sample strip on low-volume side of magnet to grab beads, 1’. Remove 2˚ antibody solution and replace with 20 µL pAG-Tn5 dilution. Do not resuspend the beads. Remove the strip from the magnet and incubate with no movement 1 hr at RT.
?. [pAG-Tn5 tethering]
Wash: place strip on low-volume side of magnet to grab beads, 1’. Remove tethering solution and replace with 20 µL 300Wash+ buffer. Do not resuspend the beads. Incubate on the magnet for 2'.
?. [chromatin tagmentation]
Prepare 300Mg+ tagmentation buffer: aliquot 180 µL 300Wash+ buffer to an eppendorf and add 20 µL 100 mM MgCl2 and mix.Store at RT. Final concentration is 10 mM MgCl2.
?. [chromatin tagmentation]
Place sample strip on low-volume side of magnet to grab beads, 1’. Remove buffer and add 20 µL 300Mg+ tagmentation buffer. Promptly move strip to a preheated PCR machine and incubate 1 hr 37˚.
?. [chromatin tagmentation]
Prepare 300STOP buffer: aliquot 200 µL 300Wash+ buffer,. Add 3.3 µL 10% SDS and 3 µL 20 mg/mL protease K. Mix by pipetting. Store at RT until tagmentation reactions are complete.
?. [chromatin tagmentation]
At the end of tagmentation, add 20 µL 300STOP buffer to each sample tube and pipette to mix. Incubate samples in a PCR machine 1 hr 58˚C.
?. [DNA recovery]
Add 60 µL 300Wash+ buffer to each sample.
?. [DNA recovery]
Work in the hood: aliquot 100 µL PCI into 8 eppendorf tubes. Transfer each sample with beads into each eppendorf, cap, and mix by full-speed vortexing ~2".
?. [Library enrichment]
Beads stick very well in this step. Aspirate off ethanol and replace with 700 µL 80% ethanol while still on magnet. Incubate 30”.
?. [Library enrichment]
Slowly aspirate to remove all traces of ethanol. Do not air-dry the beads but proceed immediately to the next step.
?. [Library enrichment]
Resuspend beads in 25 µL 10 mM Tris-HCl pH 8.
?. [Library enrichment]
Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.
?. [Sequencing & analysis]
Mix barcoded libraries to achieve equal representation as desired aiming for a final concentration as recommended by the sequencer manufacturer. After mixing, perform an Ampure bead cleanup if needed to remove any residual PCR primers.
?. [Prepare solutions and beads]
Prepare 1˚ antibody dilutions in dbe+ buffer. You’ll need 20 µL for each sample, typically at 1:100 dilution.For example, if you are doing 8 reactions with 1 antibody, aliquot 200 µL dbe+ buffer, add 2 µL 1˚ antibody, and pipette to mix. Antibody dilutions can be stored for 1 week at 4˚.
?. [Prepare solutions and beads]
Prepare ConA bead slurry:Vortex bottle of Bio-Mag Plus Concanavalin A-coated beads. Aliquot 200 µL of Bead Binding buffer to an eppendorf tube, add 45 µL of ConA beads, and pipette to mix. Incubate 5' Grab beads with a magnet for 1’, remove buffer and resuspend ConA beads in 180 µL of Binding buffer.
?. [Prepare solutions and beads]
Prepare Dig-Block-EDTA (dbe+) Buffer (1 mL):Add 1 mL of Wash+ Buffer to an eppendorf tubeAdd 33 µL 30% BSA                              final 1%Add 4 µL 0.5 M EDTA                           final 2 mMAdd 10 µL 5% Digitonin/DMSO           final 0.05%Keep on ice.
?. [DNA recovery]
Prepare 8 phase-lock tubes: spin at max, 30". Number the tubes.
?. [DNA recovery]
Transfer PCI/sample mix into a phase-lock tube. Spin at 16,000 x g, 4'.
?. [DNA recovery]
Add 100 µL chloroform to each phase-lock tube and invert 10X to mix (do not vortex). Spin at 16,000 x g, 4'.
?. [DNA recovery]
Transfer the aqueous layer by pipetting to a fresh 1.5 mL tube containing 250 µL 100% ethanol on ice. Invert tubes 10X to mix. Incubate 1' on ice.
?. [DNA recovery]
Centrifuge 10' at 16,000 x g, 4˚.
?. [DNA recovery]
Carefully pour off ethanol, and replace with 300 µL 80% ethanol. Centrifuge 4' at 16,000 x g, 4˚.
?. [DNA recovery]
Carefully pour off ethanol, and allow samples to air-dry on a paper towel, 15'.There is no visible pellet.
?. [DNA recovery]
Resuspend DNA in 22 µL 10 mM Tris-HCl, pH 8 and vortex on full to dissolve. Spin briefly to collect.
?. [Library enrichment]
Aliquot 2 µL of 10 µM Universal or barcoded i5 primer + 2 µL of 10 µM uniquely barcoded i7 primer into each tube of a PCR tube strip, using a different barcode for each sample*. Add 21 µL of sample DNA.*Indexed primers are described by Buenrostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486 (2015).
?. [Library enrichment]
Chill PCR strip on ice. Add 25 µL NEBNext HiFi 2X PCR Master mix. Vortex and spin briefly to mix, and return to ice.Do not use hot-start enzyme mixes.
?. [Library enrichment]
Start cycling program on thermocycler  to get to temperature. When the block gets to 58˚ move the tube strip into the machine.Heated lidCycle 1: 58˚ for 5' (gap filling)Cycle 2: 72˚ for 5' (gap filling)Cycle 3: 98˚ for 30"Cycle 4: 98˚ for 10"Cycle 5: 60˚ for 10"Repeat Cycles 4-5 13 times72˚ for 1' and hold at 8˚This typically takes 30' to run.To minimize the contribution of large DNA fragments and excess primers, PCR should be performed for 12-14 cycles, preferably with a 10" 60-63˚ combined annealing/extension step.Do not add extra PCR cycles to see a signal by capillary gel electrophoresis (eg. Tapestation). Observing no signal for a sparse chromatin protein such as a transcription factor is normal, and the barcoded sample can be concentrated for mixing with the pool of barcoded samples for sequencing. Extra PCR cycles reduce the complexity of the library and may result in an unacceptable level of PCR duplicates.
?. [Library enrichment]
Label eppendorfs with library ID and aliquot 65 µL Ampure XP beads to each tube. This is a 1.3:1 ratio for cleanup.
?. [Library enrichment]
Transfer PCR reactions to eppendorfs with Ampure beads and vortex to mix. Quickly spin the tubes to collect the beads, and incubate 5' RT.
?. [Library enrichment]
Place eppendorfs on magnet and incubate 5' to grab beads.
?. [Library enrichment]
Beads are somewhat slippery in this step. Pipette off solution and replace with 700 µL 80% ethanol while still on magnet. Incubate 30".
?. [Sequencing & analysis]
Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. PE25 is more than sufficient for mapping to the Drosophila genome.
?. [Sequencing & analysis]
Mapping, spike-in normalization and analysis are described here:dx.doi.org/10.17504/protocols.io.bjk2kkye
?. [Prepare solutions and beads]
Prepare 300Wash+ Buffer (15 mL):Add 15 mL of 300Wash $ buffer solution to a 15 mL conical tube Add 1 mini Roche cOmplete EDTA-free tabletsAdd 3.8 µL 2 M Spermidine             final 0.5 mMStore overnight at 4˚C.