Feb 24, 2026
Pico-C for Drosophila Embryos
- Noura Maziak1,2,
- Vaquerizas Lab3
- 1MRC Laboratory of Medical Sciences, London, W12 0HS, UK;
- 2Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0HS, UK;
- 3http://www.vaquerizaslab.org
Protocol Citation: Noura Maziak, Vaquerizas Lab 2026. Pico-C for Drosophila Embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31nm1l25/v1
Manuscript citation:
Maziak N, Zhang Y, Groll F, Brown HE, Madich A, Kaur Y, Harrison MM, Zhou J, Vaquerizas JM. 3D Genome Reorganization Foreshadows Zygotic Genome Activation in Drosophila. Nature Genetics. In press.
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: November 05, 2025
Last Modified: February 24, 2026
Protocol Integer ID: 231520
Keywords: drosophila embryos pico, quality contact maps in drosophila, drosophila, nc14 embryo, nuclei, pico, input micro, magnetic bead, coated magnetic bead, quality contact map, micro
Funders Acknowledgements:
Medical Research Council, UK
Grant ID: MC_UP_1605/10
Academy of Medical Sciences and the Department of Business, Energy and Industrial Strategy
Grant ID: APR3\1017
Abstract
Pico-C is a low-input Micro-C method that enables the generation of high-quality contact maps in Drosophila using as few as 10 NC14 embryos (~60,000 nuclei) per experiment. It employs Con A–coated magnetic beads to bind nuclei and minimize sample loss, a strategy inspired by ChIC, CUT&RUN, and similar approaches.
Guidelines
We find Con A–coated magnetic beads to be very “sticky” when using pre-lubricated tubes. To minimize nonspecific adsorption and sample loss, perform all steps involving nuclei in Eppendorf Protein LoBind 1.5 mL PCR-clean tubes (Eppendorf, E0030108116). For steps at and after DNA extraction, switch to Eppendorf DNA LoBind tubes (Eppendorf, 0030108051).
We avoid vortexing when nuclei are bound to Con A–coated beads to keep the nuclei intact and minimize sample loss. We also limit mixing by pipetting as much as possible throughout the whole protocol—most mixing is instead performed manually by flicking of the tubes.
Materials
Buffers (buffers in bold are prepared partially or entirely fresh):
- Buffer #1 (MB1): 50 mM NaCl, 10mM Tris-HCl pH 7.5, 5mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1X protease inhibitors
- Buffer #2 (MB2): 50 mM NaCl, 10mM Tris-HCl pH 7.5, 10mM MgCl2
- Buffer #3 (MB3): 50 mM Tris-HCl pH 7.5, 10mM MgCl2
- 10X Binding Buffer (for Concanavalin A-coated magnetic beads): 200 mM HEPES-KOH pH 8, 100 mM KCl, 10 mM CaCl₂, 10 mM MnCl₂, 5 mM spermidine
- 1X Binding Buffer (for Concanavalin A-coated magnetic beads): 20 mM HEPES-KOH pH 8, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂, 0.5 mM spermidine, 1X protease inhibitors.
- TE buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA
- 2X Bind and Wash buffer (2X BW; for Streptavidin magnetic beads): 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl
- 1X Bind and Wash buffer with 0.1% Triton (TBW; for Streptavidin magnetic beads): 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.1% Triton X-100
- Extraction buffer: 50 mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM EDTA, 1% SDS
General:
- cOmplete‱ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail; Roche 5892791001
- 10% NP-40 Substitute, ab142227
- Magnetic Rack for 1.7 mL tubes
- 1M MnCl₂; Manganese(II) chloride, 1M aq. soln. Alfa Aesar J63150
- Triton X-100
- HEPES, 0.5M buffer soln., pH 8.0; Alfa Aesar J63002.AE
- 2M Tris-HCl pH 7.5
- 1M Tris-HCl, pH 8.0
- 2M MgCl2 ; Magnesium Chloride 2M Solution, Fisher Scientific 50-997-920
- Eppendorf Protein LoBind 1.5 mL PCR-clean tubes; Eppendorf, E0030108116
- Eppendorf DNA LoBind tubes; Eppendorf, 0030108051
- 100% Ethanol
Digestion:
- BP531 BioMag Plus Concanavalin A
- MNase (micrococcal nuclease) Worthington Biochem LS004798
- 0.5M EGTA; Thermo scientific 15425795
- RNase A
DNA Extraction:
- Phenol:Chloroform:Isoamylic alcohol (PCIa) Sigma-Aldrich P2069
- GlycoBlue (15mg/mL); Thermo Scientific AM9515
Repair fragment ends and ligation :
- ATP Solution (100mM); Themo Scientific R0441
- DTT 100mM; Promega P1171
- NEBuffer 2.1; New England Biolabs (NEB) B7202S
- T4 Polynucleotide Kinase (T4 PNK); New England Biolabs (NEB) M0201
- T4 DNA Ligase; New England Biolabs (NEB) M0202
- DNA Polymerase I, Large (Klenow) Fragment; New England Biolabs (NEB) M0210S/L
- DNA Polymerase I, Large (Klenow) Fragment; New England Biolabs (NEB) M0210M (more concentrated- for spike in during end-labeling)
- Biotin-14-dATP; Jena Biosciences #NU-835-BIO14
- Biotin-11-dCTP; Jena Bioscience #NU-809-BIOX
- dTTP and dGTP
- 20 mg/ml BSA
- T4 DNA Ligase Reaction Buffer; New England Biolabs (NEB) B0202S (will also come with T4 DNA Ligase)
- EDTA solution pH 8.0 (0.5 M) for molecular biology; Panreac AppliChem A4892,0500
Library Preparation:
- Dynabeads MyOne Streptavidin C1 Invitrogen #65001 or Dynabeads MyOne Streptavidin T1 Invitrogen #65601 (Note: although similar, Goel et al. 2023; PMID: 37157000; found that T1 beads outperformed by 1.5-3 fold which has led us to switch)
- NEBNext Ultra II DNA Library Prep Kit; New England Biolabs #E7645 (Note: We use KAPA HIFI for last PCR step instead of the NEB Q5 that comes with the kit)
- KAPA HiFi HotStart ReadyMix; KAPA Biosystems #KK2601
- AMPure XP Beads for DNA Cleanup; Beckman Coulter A63882
Troubleshooting
Before start
It’s a good idea to read a few steps ahead in this protocol, as this protocol involves multiple temperature changes and long incubations.
Pre-preparation (Buffers and Concanavalin A Magnetic Bead clean-up)
Freshly prepare MB1, store on ice
| Components | For 10 mL | Final | |
| 5M NaCl | 100 μL | 50 mM NaCl | |
| 2M Tris-HCl (pH 7.5) | 50 μL | 10 mM Tris-HCl | |
| 2M MgCl2 | 25 μL | 5 mM MgCl2 | |
| 1M CaCl2 | 10 μL | 1 mM CaCl2 | |
| 10% NP-40 | 200 μL | 0.2% NP-40 | |
| H2O | 9.615 mL | ||
| Protease Inhibitor | 1 tablet (Roche cOmplete ULTRA Tablets Mini EDTA-free) | 1X |
MB1 stock can be pre-prepared excluding NP-40 and PIC which should be added freshly.
Prepare MB2, store on ice. (Not required if performing only the MNase Digestion Test)
| Components | For 10 mL | Final | |
| 5M NaCl | 100 μL | 50 mM NaCl | |
| 2M Tris-HCl (pH 7.5) | 50 μL | 10 mM Tris-HCl | |
| 2M MgCl2 | 50 μL | 10 mM MgCl2 | |
| H2O | 9.8 mL |
We usually pre-prepare a large stock of MB2 and store at 4°C.
Prepare MB3, store on ice. (Not required if performing only the MNase Digestion Test)
| Components | For 10 mL | Final | |
| 2M Tris-HCl (pH 7.5) | 250 μL | 50 mM Tris-HCl | |
| 2M MgCl2 | 50 μL | 10 mM MgCl2 | |
| H20 | 9.7 mL |
We usually pre-prepare a large stock of MB3 and store at 4°C.
Freshly prepare 10X and 1X Binding Buffer
| 10X Binding Buffer Components | For 4 mL | Final | |
| 0.5 M HEPES-KOH (pH 8) | 1.6 mL | 200 mM HEPES-KOH | |
| 1M KCl | 400 μL | 100 mM KCl | |
| 1M CaCl2 | 40 μL | 10 mM CaCl2 | |
| 1M MnCl2 | 40 μL | 10 mM MnCl2 | |
| 1M spermidine | 20 μL | 5 mM spermidine | |
| H20 | 1.9 mL |
We make this freshly. Be sure not to confuse MnCl2 needed here with MgCl2 used in MB buffers.
| 1X Binding Buffer Components | For 10 mL | Final | |
| 10X Binding Buffer | 1 mL | 1X | |
| Proetease Inhibitor | 1 tablet (Roche cOmplete ULTRA Tablets Mini EDTA-free) | 1X | |
| H2O | 9 mL |
We pre-dissolve the Protease Inhibitor tablet at room temperature in water @ rotation before adding 10X Binding Buffer since this can take a while.
Prepare Concanavalin A Magnetic Beads in non-pre-lubricated tubes (we tend to use Eppendorf Protein LoBind 1.5 mL PCR-clean tubes #E0030108116).
Gently resupend and withdraw enough bead slurry such as there is 10 μL for each final sample in a 2 mL tube.
To wash, add 1.5 mL 1X Binding Buffer, mix by inversion and quick pulse centrifuge to remove any liquid from cap.
Place tube on magnet stand to clear (30s to 2 min). Remove liquid.
Repeat wash and remove liquid.
Resuspend in a volume of 1X Binding Buffer equal to volume of initial bead slurry (10 μL per sample).
Required only if performing MNase Digestion Test:
Prepare Extraction Buffer
| Component | For 100 mL | Final | |
| 1M Tris-HCl, pH 8 | 5 mL | 50 mM Tris-HCl, pH 8.0, 50mM NaCl, 1mM EDTA, 1% SDS | |
| 5M NaCl | 1 mL | ||
| 0.5M EDTA | 0.2 mL (200 µL) | ||
| 20% SDS | 5 mL | ||
| H2O | 88.8 mL |
Can be stored away from light at room temperature
Day 1: Chromatin digestion (part 1)
Resuspend the fixed, flash-frozen embryos in 500 µL in cold complete MB1 buffer.
Working quickly, crush with cold metal pellet pestle until the solution is clear, avoiding bubbles.
Incubate for 20 minutes on ice.
Centrifuge for 5 min at 5000xg at 4°C. Discard supernatant.
Remove supernatant and add 500 µL of complete MB1. Mix by gently flicking the tube.
Centrifuge for 5 min at 5000xg at 4°C. Discard supernatant.
Resuspend in 900 μL MB1 by flicking the tube.
Add 100 μL of 10X binding buffer to nuclei to a 1X concentration.
Add the Con-A bead slurry prepared previously (10 μL per sample).
Rotate 10 min at room temperature.
Place samples on magnet, remove liquidm and wash nuclei/beads in 500 µL of cold complete MB1 once. Place on magnet and remove liquid.
Resuspend in 500 μL of cold complete MB1. Place samples on ice.
Working quickly and on ice, add the appropriate amount of MNase to each sample to digest chromatin, as determined by the digestion test and mix well by flicking. If performing an MNase Digestion Test, include multiple MNase concentrations to capture a range of digestion efficiencies.
Incubate for 10 min at 37oC with shaking at 950 rpm.
Add 4 μL of 0.5M EGTA for a final concentration at 4 mM to stop reaction (flick tube to mix).
Place samples on magnet and remove supernatant.
MNase Digestion Test64 steps
If performing only an MNase Digestion Test, wash the sample once with MB2 and then proceed directly to the attached protocol.
Note: Although we did not observe variability in MNase efficiency across different fixation batches (even when comparing samples fixed on different continents), MNase itself can vary between enzyme batches. Therefore, if switching to a new MNase source, a fresh digestion test should be performed.
MNase_Digestion.pdf
549KB
Keep samples on magnet for wash and add 500 μL of cold MB2 without dislodging beads. Remove liquid (while samples remain on magnet stand).
Repeat wash 3 times.
Discard supernatant and proceed to Repair fragment ends section (samples can be removed from magnetic stand now and placed on ice).
Day 1: Repair fragment ends (part 2)
Add 45 µL of master-mix (below) to each sample.
| A | B | C | |
| 100 mM ATP | 1 μL | 2mM ATP | |
| 100 mM DTT | 2.5 μL | 5 mM DTT | |
| 10X NEBuffer 2.1 | 5 μL | 50mM NaCl, 10mM Tris, 10mM MgCl2, 100ug/ml BSA | |
| 10U/uL T4 PNK | 2.5 μL | ~10U/1ug DNA (NEB End repair module: 5U for 1 ug DNA) | |
| H2O | 34 μL |
Incubate for 15 min at 37ºC (we do this with 800 rpm interval mixing).
Add 5 uL of 5U/μL NEB Klenow Fragment and continue incubation at 37ºC for an additional 15 min. Do not discard supernatant after completing this step.
Prepare the end-labeling master mix as described below, then add 25 µL per sample, bringing the final volume to 75 µL.
| Component | For 75 uL total (one sample) | Final | |
| 1mM Biotin-dATP (14; JenaBio) | 5 µL | 66 µM dNTP/each 1.67mM ATP 100 ug/mL BSA 33 mM NaCl, 23 mM Tris, 10 mM MgCl2. 6.67 mM DTT | |
| 1mM Biotin-dCTP (11; JenaBio) | 5 µL | ||
| 10 mM dTTP + dGTP | 0.5 µL | ||
| 20 mg/ml BSA (200X) | 0.125 µL | ||
| 10X T4 DNA Ligase Buffer | 2.5 µL | ||
| H2O | 11.875 |
It is best to prepare a master-mixes for all samples at once, using 1.5X the total number of reactions to ensure sufficient volume.
Incubate for 2 hours at room temperature (25ºC) with 800 rpm interval mixing.
Add 1.5 µL of 100 mM ATP and 1 µL of Klenow Fragment (50 U/µL) to the reaction, then continue incubating for 2 hours at room temperature (25 °C) with interval mixing at 800 rpm.
Add 4.5 µL 0.5 M EDTA (for a final concentration of 30 mM). Mix by flicking.
Place samples on the magnetic stand and leave them on the magnet for the entire wash step. Add 500 µL of cold MB3, do not dislodge beads. Remove liquid. Repeat wash again once.
Day 1: Proximity ligation, biotin cleanup, and overnight reverse crosslinking (part 3)
Add 250 µL of the ligation master-mix below to each sample.
| Component | For 250 µL | Final | |
| H2O | 221.25 µL | 50 mM Tris, 10mM MgCl2, 1mM ATP, 10mM DTT, 100 ug/mL BSA | |
| 10X DNA Ligase Buffer | 25 µL | ||
| 20 mg/mL BSA (200X) | 1.25 µL | ||
| 2000U/ µL T4 DNA Ligase | 2.5 µL | 5000 U |
Incubate for 2.5 hours at room temperature with slow rotation.
Add an additional 2.5 µL of 2000U/µL T4 DNA Ligase (for a total of 10,000 U) and continue
incubation for another 2.5 hours with rotation.
Place samples on magnet, and discard supernatant.
Incubate each sample with the master mix (see below) for 15 minutes at 37 °C, using interval mixing at 850–950 rpm.
| Component | For 100 µL | Final | |
| 10X NEBuffer #1 | 10 µL | 10mM Bis-Tris-Propane-HCl, 10mM MgCl2,1mM DTT | |
| H2O | 85 µL | ||
| 100U/µL Exonuclease III | 5 µL | 500 U |
Add 12.5 µL of 20 mg/mL Proteinase K, 12.5 µL of 10% SDS, and 5.2 µL of 5 M NaCl to each sample, then incubate at 65 °C overnight.
Day 2: DNA purification
For phenol:chloroform:isoamyl alcohol (PCIa) extraction, add 1X sample volume (125 µL) of PCIa. Beads will remain in the mixture.
Vortex for 20 seconds, then let the samples rest for 2–3 minutes (or until the phases separate). This pause helps the beads settle at the bottom during centrifugation.
Centrifuge for 10 minutes at 19,800 × g at room temperature, then carefully collect and retain the upper aqueous phase.
Add 0.1X volume sodium acetate (12.5 µL), 2.5X volume of ice-cold 100% ethanol (312.5 µL), and 1 µL glycogen to the sample. Shake to mix.
Incubate for at least 15 minutes at −80°C to allow DNA precipitation. This step can serve as a pause point; samples may be stored at −80 °C indefinitely.
Optional stopping point
Wash pellet with cold 75% ethanol. Do not mix.
Spin for 5 min at max speed in ultracentrifuge at 4°C.
Remove ethanol, do not contact pellet with pipette.
Air dry pellet for 10 min at room temperature.
Resuspend the pellet in 50 µL of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA) supplemented with 1X RNase A.
Incubate for 15 minutes at 37°C.
Quantify DNA by Qubit.
Day 2: Library Preparation
Prepare 2X Bind & Wash Buffer (BW; 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) and 1X Bind & Wash Buffer with 0.1% Triton (TBW).
Wash 5 µL magnetic streptavidin beads per sample with 1X Bind & Wash buffer + 0.1% Triton (TBW).
Place on magnet, remove TBW, and resuspend beads in 150 µL 2X BW buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) per sample.
Dilute DNA sample in H2O to reach 150 µL and add 150 µL of beads in 2X BW buffer.
Rotate for 20 min at room temperature. Then place on magnet and remove supernatant.
Add 400 µL 1× TBW and mix at 55°C for 2 min with interval mixing. Place on magnet and remove liquid. Repeat again once.
Add samples on magnet, remove liquid and 10 mM Tris pH 7.5 (100-400 µL). Place on magnet and remove supernatant.
Resuspend beads in 25 µL 10 mM Tris pH 7.5.
Add 3.5 µL of NEBNext Ultra II End Prep Reaction Buffer and 1.5 µL of NEBNext Ultra II End Prep Enzyme Mix to each sample and mix gently by flicking. Your per sample volume should be 30 µL now.
Incubate for 30 min at 20°C with interval mixing at 850 rpm, followed by 30 min at 65°C.
Put samples on ice and add add 0.5 µL of Adaptor for Illumina, 15 µL NEBNext Ultra II Ligation Master Mix, and 0.5 µL of Ligation Enhancer. Mix thoroughly by flicking and incubate for 30 min @ 20°C with interval mixing at 850 rpm.
Add 1.5 µL of USER enzyme, mix samples thoroughly by flicking, and incubate at 37°C with interval mixing at 850 rpm.
Wash the beads with 1X TBW buffer by incubating at 55°C with interval mixing. Place the tube on a magnet to separate the beads and discard the supernatant.
Next, wash the beads with 10 mM Tris buffer pH 7.5 (100-400 µL). Place the tube on the magnet again, discard the supernatant, then resuspend the beads in 50 µL of 10 mM Tris buffer pH 7.5. Keep the beads on ice until further use.
(Optional) Pilot PCR - We do this if dealing with new sample-types, to asses the number of PCR cycles needed for library prep using 20% of the sample. This is a good step to incorporate early on when establishing the protocol to grasp how samples behave and avoid potential over-amplification.
For this create the following master-mix:
| Component | For 50 µL | |
| Streptavidin beads in Tris | 10 µL | |
| Nuclease-free water | 14 µL | |
| 2X KAPA HiFi Hot Start Mix | 25 µL | |
| 10uM universal primer | 0.5 µL | |
| 10uM index primer | 0.5 µL |
Mix the contents thoroughly, then briefly centrifuge the tubes to collect the PCR reactions at the bottom.
Then run PCR as follows:
| Step | Temperature | Time | |
| Denaturation | 98°C | 45 sec | |
| 8-14 Cycles | 98°C | 15 sec | |
| 60°C | 30 sec | ||
| 72°C | 30 sec | ||
| Extension | 72°C | 1 min | |
| Hold | 4°C | ∞ |
After 8-10 cycles, pause the PCR at the end of the extension step and take out 10 µL, then restart the PCR
and continue for another two cycles. Do this four to five times, e.g. cycles 8, 10, 12,
14, or cycles 10, 12, 14, 16 if you expect a low concentration input.
You can take 1 µL of each sample to Qubit, and mix the rest with DNA dye to run on a 2% agarose TAE gel (you can directly mix the beads with loading dye and load them, the beads do not affect the gel).
At higher cycle numbers, you should see bands in the expected di-nucleosomal and tri-nucleosomal
ligation product + adaptors size range (N.B. adaptors + indexes are ~120bp). Use this to determine how many PCR cycles to use for the final library prep PCR. We have also used Qubit concentrations can also be used as a reference.
For final PCR, set up 2 or 4 parallel PCRs for each sample as follows on ice. If no pilot PCR was carried-out, adjust streptavidin bead volume and water volume accordingly.
| Component | 4 parallel PCRs per sample | 2 parallel PCRs per sample | |
| 10uM universal primer | 0.5 µL | 0.5 µL | |
| 10uM index primer | 0.5 µL | 0.5 µL | |
| Streptavidin beads in Tris | 10 µL | 20 µL | |
| Nuclease-free water | 14 µL | 4 µL | |
| 2X KAPA HiFi Hot Start Mix | 25 µL | 25 µL |
Mix the contents thoroughly, then briefly centrifuge the tubes to collect the PCR reactions at the bottom.
Run PCR as shown below. Note that if more than 12 cycles are necessary, the library may have high
levels of PCR duplicates.
| Step | Temperature | Time | |
| Denaturation | 98°C | 45 sec | |
| 8-12 Cycles | 98°C | 15 sec | |
| 60°C | 30 sec | ||
| 72°C | 30 sec | ||
| Extension | 72°C | 1 min | |
| Hold | 4°C | ∞ |
Optional stopping point
Day 2: Library size-selection (two-sided)
Bring AMPure XP or SPRI beads to room temperature for 30 min.
Note: volumes and proper pipetting are very important for these steps. Carry steps below at room temperature unless noted otherwise.
Make fresh 80% ethanol.
Transfer the PCR supernatant to a new tube, pooling all PCR reactions for the same sample. Adjust the total volume to 200 µL with water.
If desired, remove 1 µL for Bioanalyzer analysis and 1 µL for Qubit quantification; if you do, add 2 µL of water back to maintain the final volume.
For upper cutoff selection, add exactly 0.55X volumes (110 μL) of Ampure beads. Mix thoroughly by flicking, shaking, or using a low-speed vortex, and allow the mixture to sit for 5-10 minutes.
Separate on magnetic stand (allow 5 min or so to let the beads get settled).
Move supernatant to a new tube. This is your library, do not discard.
Add 0.2X volumes (40 μL) of Ampure beads (this yields to a total of 0.75X volumes of Ampure buffer in the tube). Mix thoroughly by flicking, shaking, or using a low-speed vortex, and allow the mixture to sit for 5-10 minutes.
Separate on magnetic stand and discard supernatant. In this step, your library is on the beads.
Add 700 μl of 80% ethanol to the tube on the stand, taking care not to disturb the pellet, and let sit for 30 sec. Remove ethanol and repeat wash. Remove ethanol completely.
Let samples dry for a max. of 5 min. Over-drying can lead to DNA-loss.
Resuspend beads in 25-30 μL 10 mM Tris pH 8.0 and incubate at 37°C for 5 min.
In case of issue with primer-dimer contamination.4 steps
The above cleanup should be sufficient at removing primer-dimer contamination from PCR. In case that is not the case, you can follow the following protocol to its end. This includes an additional, now single-sided AMPure cleanup at 0.8X ratio. Should be avoided if not needed.
Separate on magnetic stand and move supernatant containing the final library to a fresh tube. This is your final library!
Library QC
Measure concentration using Qubit. We use DNA HS for this.
Measure average fragment size using Bioanalyzer.
We often perform a shallow sequencing run. In our experience, this has not been very useful for predicting duplication rates/library complexity (despite testing multiple tools), but it does allow extraction of key library metrics, most importantly the cis-fraction (which can be obtained using pairtools). This value can be an important indicator of library quality: high-quality Micro-C maps typically show >80% of contacts in cis. For Drosophila embryos, we consistently observe ~95% cis-fraction.
Protocol references
Blythe, S. A., & Wieschaus, E. F. (2016). Establishment and maintenance of heritable chromatin structure during early Drosophila embryogenesis. eLife, 5, e20148. https://doi.org/10.7554/eLife.20148
Hsieh, TH., Fudenberg, G., Goloborodko, A. et al. Micro-C XL: assaying chromosome conformation from the nucleosome to the entire genome. Nat Methods 13, 1009–1011 (2016). https://doi.org/10.1038/nmeth.4025
Skene, P. J., & Henikoff, S. (2017). An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife, 6, e21856. https://doi.org/10.7554/eLife.21856
Hsieh, T. H. S., et al. (2020). Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Molecular Cell, 78(3), 539–553. https://doi.org/10.1016/j.molcel.2020.03.002
Ing-Simmons, E., Vaid, R., Bing, X. Y., et al. (2021). Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nature Genetics, 53, 487–499. https://doi.org/10.1038/s41588-021-00799-x
Goel, V.Y., Huseyin, M.K. & Hansen, A.S. Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments. Nat Genet 55, 1048–1056 (2023). https://doi.org/10.1038/s41588-023-01391-1