TITLE: CUT&RUN with Drosophila tissues
AUTHORS: Kami Ahmad

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We have modified the Cleavage Under Targets and Release Using Nuclease (CUT&RUN) method for epigenomic profiling of histone modifications and chromatin proteins to use dissected Drosophila tissues. In CUT&RUN, cells or tissues are permeabilized and incubated under conditions where a factor-specific antibody can bind at sites of a chromatin protein. This antibody is then used to tether a protein A-micrococcal nuclease fusion protein, which upon activation releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing, and leaving the vast majority of DNA behind in the cellular pellet. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. Previous versions of CUT&RUN have used isolated nuclei, mammalian cultured cells, or yeast spheroplasts. Here we have modified the CUT&RUN protocol to manipulate imaginal discs and tissues dissected from larvae with magnetic beads without isolation of cells or nuclei. About 10 imaginal discs provides high-quality data for a histone modification or for a chromatin factor. A simple spike-in strategy is used for accurate quantitation of experiment yields and library preparation. From larvae to purified DNA, CUT&RUN requires 1-2 days with minimal handling after dissection.</div></div>

[STEPS]
?. [Prepare solutions and beads]
Prepare a fresh 5% digitonin solution as follows:Weigh out 50 mg digitonin powder in a 2 ml microcentrifuge tube. Boil some water in a small beaker in a microwave oven, and pipette in and out to warm the 1000 μL pipette tip. Pipette the hot water into the tube with the digitonin powder, close the cap, and quickly vortex on full until the digitonin is completely dissolved. If refrigerated, this stock can be used within a week, but will need reheating as the digitonin slowly precipitates. The effectiveness of digitonin varies between batches, so testing permeability of Trypan blue is recommended to determine effective concentrations. We have obtained excellent results with 0.02-0.1% digitonin.
• CAUTION: Digitonin is toxic and care should be taken especially when weighing out the powder.
?. [Prepare solutions and beads]
Prepare Wash+ Buffer (50 mL):Add 50 mL of wash buffer stock solution to a 50 mL conical tube Add 1 large Roche complete EDTA-free tabletsAdd 125 µL 200 mM Spermidine     final 0.5 mMKeep on ice or store overnight at 4˚C.
?. [Prepare solutions and beads]
Prepare Dig-Block-EDTA (dbe+) Buffer (25 mL):Add 25 mL of Wash+ Buffer to a 50 mL conical tubeAdd 100 µL 0.5 M EDTA                       final 2 mMAdd 250 µL 5% Digitonin                     final 0.05%Keep on ice or store overnight at 4˚C.
?. [Prepare solutions and beads]
Prepare ConA beads:Gently vortex bottle of Bio-Mag Plus Concanavalin A-coated beads. Transfer 150 µL of ConA bead slurry to an eppendorf. Grab beads with a magnet for 5’, remove buffer and replace with 1 mL Binding buffer. Incubate 1’. Wash 2X. Resuspend ConA beads in 150 µL of Binding buffer.
?. [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 2 wells of a clean shallow dissection dish for each sample. Pick up larvae and transfer to the dissection dish, and dissect out 10 wing discs. Aim to take less than 10 minutes, so that discs remain healthy. Use a 200 µL pipette tip to transfer the wing discs to the second well of Wash+ buffer.
?. [Binding tissues to beads]
Add 15 µL ConA bead suspension to an eppendorf. Use a 200 µL pipette tip to transfer the wing discs to the eppendorf with a minimal volume of Wash+ buffer. Mix by gentle pipetting. 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.
?. [Binding primary antibody]
Block the tissues: Add 1 mL dbe+ buffer to each eppendorf. Gently flick the tube to mix, and incubate 10’ at RT.
?. [Binding primary antibody]
Prepare antibody dilutions in dbe+ buffer. You’ll need 100 µL for each sample, typically at 1:100 dilution.For example, you are doing 10 reactions with 1 antibody. Aliquot 1 mL dbe+ buffer, add 10 µL 1˚ antibody, and pipette to mix. If you are doing 10 different antibodies, aliquot 100 µL dbe+, add 1 µL 1˚ antibody and mix.
?. [Binding primary antibody]
Place eppendorfs on magnet, and let bind for 2’. Pipette off the buffer, and replace with 100 µL antibody dilution. Incubate eppendorfs at a 60˚ angle on an orbital shaker, orbiting slowly, 4˚C O/N.
?. [Binding primary antibody]
Very briefly spin tubes to collect liquid if necessary. Place tubes on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL dbe+ buffer to wash the beads. Resuspend sample by pipetting and incubate 2’ RT.
?. [Binding primary antibody]
Repeat Wash. Very briefly spin tubes to collect liquid if necessary. Place tubes on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL dbe+ buffer to wash the beads. Resuspend sample by pipetting and incubate 2’ RT.
?. [Optional: Binding secondary antibody]
This section uses a secondary antibody to adapt mouse IgG primary antibody for protein-A binding. If not needed, skip to Step 17.Prepare secondary rabbit anti-mouse IgG antibody dilution in dbe+ buffer. You’ll need 100 µL for each sample at 1:100 dilution. For 10 reactions with a mouse primary antibody, aliquot 1 mL dbe+ buffer, add 10 µL 1˚ antibody, and pipette to mix.
?. [Optional: Binding secondary antibody]
Place sample eppendorfs on magnet, and let bind for 2’. Pipette off the buffer, and replace with 100 µL secondary antibody dilution. Incubate eppendorfs at a 60˚ angle on an orbital shaker, orbiting slowly, 1 hr at RT.
?. [Optional: Binding secondary antibody]
Very briefly spin tubes to collect liquid if necessary. Place tubes on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL dbe+ buffer to wash the beads. Resuspend sample by pipetting and incubate 2’ RT.
?. [Optional: Binding secondary antibody]
Repeat Wash. Very briefly spin tubes to collect liquid if necessary. Place tubes on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL dbe+ buffer to wash the beads. Resuspend sample by pipetting and incubate 2’ RT.
?. [nuclease tethering]
Dilution of different batches of pAMN are calibrated. This step uses Batch #6, which is used at a 1:400 dilution. Use the appropriate calibrated dilution of other batches.Prepare pAMN dilution: Aliquot 1 mL dbe+ buffer, add 2.5 µl pAMN#6 and mix by pipetting. This is enough for 10 samples.
?. [nuclease tethering]
Place sample tubes on magnet to grab beads, 2’. Remove buffer, and replace with 100 µL pAMN dilution. Resuspend sample by pipetting and incubate orbiting slowly 1 hr, RT.
?. [nuclease tethering]
Wash: very briefly spin tubes to collect liquid if necessary. Place samples on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL Wash+ buffer. Resuspend samples by pipetting and incubate 2’.
?. [nuclease tethering]
Repeat wash: very briefly spin tubes to collect liquid if necessary. Place samples on magnet to grab beads, 2’. Remove buffer, and replace with 500 µL Wash+ buffer. Resuspend samples by pipetting and incubate 2’.
?. [DNA cleavage]
Prepare Wash+C buffer: Aliquot 1.5 mL Wash+ buffer, add 30 µL 100 mM CaCl2 and mix. Place buffer on ice and chill. Final concentration is 2 mM CaCl2.
?. [DNA cleavage]
Place sample tubes on magnet to grab beads, 2’. Remove buffer, and place samples on ice to chill tubes. Promptly resuspend sample with 150 µl Wash+C buffer and start timer. Incubate 30’ on ice. Occasionally flick the tube to resuspend the beads.
?. [DNA cleavage]
Prepare 2XSTOPyR buffer: aliquot 1600 µL 2XSTOP buffer. Add 3.2 µL 1 ng/mL yeast spike-in DNA and 10 µL RNaseA. Chill on ice until cleavage reaction is complete.
?. [DNA cleavage]
Prepare Pellet buffer: Aliquot 800 µL Wash+ buffer, add 800 µL 2XSTOP buffer. Add 3.2 µL of 1 ng/mL yeast spike-in DNA. Add 17 µL 10% SDS and 22 µL of 20mg/mL proteinase K. Store at RT.
?. [DNA cleavage]
At the end of 30’ incubation, add 150 µL 2XSTOPyR buffer to each sample tube and pipette to mix. Incubate samples in a 37˚C water bath, 30’.
?. [DNA recovery]
This section includes DNA recovery from both supernatant and pellet fractions. In initial experiments with a new antibody, we compare the abundance of cleaved DNA in supernatant and pellet fractions to confirm that most of the cleaved DNA is soluble. in most cases this is the case, and then library construction and sequencing is performed only with supernatant DNA.The following steps describe DNA recovery from both supernatant and pellet fractions.
?. [DNA recovery]
Add 2 µL 10% SDS and 2.5 µL of 20 mg /mL Proteinase K to each ‘S’ sample and mix by brief vortexing. Incubate ‘S’ and ‘P’ samples in a 50˚C water bath, 2 hr.
?. [DNA recovery]
To ‘S’ tubes: Use wide-bore green tips to add 40 µL AmpureXP bead slurry and 560 µL MXP buffer to each tube, mix thoroughly by 10X pipetting. Incubate 15’ RT. This is a non-size-selective recovery of DNA fragments; the PEG:sample ratio is 2V. Total volume is ~900 µL.
?. [DNA recovery]
To ‘P’ tubes: grab ConA beads with magnet, 2’. Transfer supernatant to a new tube. Beads are very slippery in this buffer. Use wide-bore green tips to add 40 µL AmpureXP bead slurry and 65 µL HXP buffer to each tube, mix thoroughly by 10X pipetting. Incubate 15’ RT.This is a size-selective recovery of DNA; the PEG:sample ratio is 0.7V. Total volume is ~255 µL. The fragments you are looking for will be in the supernatant!
?. [DNA recovery]
To ‘P’ samples: grab Ampure beads with magnet, 5’. Beads are very slippery in this buffer. Transfer supernatant to a new tube, tag-labeled as ‘sample#PX’. Add 40 µL AmpureXP bead slurry and 155 µL MXP buffer to each tube, mix thoroughly by 10X pipetting. Incubate 15’ RT. This is a non-size-selective recovery of DNA; the PEG:sample ratio is 2V. Total volume is ~450 µL.
?. [DNA recovery]
24) To ‘S’ and ‘PX’ tubes: grab Ampure beads with magnet, 5’. Beads are very slippery in this buffer. Pipette off buffer, making sure not to remove beads at the end. Keep tubes on the magnet, and add 1 mL 80% ethanol to each tube. Incubate 30”.
?. [DNA recovery]
Beads stick very well in this step. Aspirate off ethanol and replace with 1 mL 80% ethanol. Incubate 30”.
?. [DNA recovery]
Slowly aspirate to remove all traces of ethanol. Leave tubes on magnet and allow beads to air-dry, 15’.
?. [DNA recovery]
Resuspend beads in 40 µL 10 mM Tris pH8.
?. [DNA recovery]
Chromatin fractionation: Grab beads on magnet, 2’. Transfer the supernatant containing cleaved chromatin particles to a new tube, marked ‘sample#S’. Resuspend pellet in 150 µL Pellet buffer, add a ‘P’ to the tube label. The 'P' tube is a transient tube.
?. [DNA recovery]
Determine the size distribution of DNA in samples by Agilent 4200 TapeStation analysis.The positive control (anti-H3K27me3) should show a nucleosomal ladder in the 'S' sample, and a weak high-molecular weight DNA band in the 'PX' sample.The negative control (non-specific rabbit IgG) should show no DNA in the 'S' sample, and a weak high-molecular weight DNA band in the 'PX' sample.Most antibodies (eg to transcription factors) will show no nucleosomal ladder, but we find that they often have sufficient material for library construction and produce excellent genomic profiles.See Guidelines for more discussion.
?. [DNA recovery]
Quantify library yield using dsDNA-specific assay, such as Qubit.