MicroC library preparation from Drosophila embryos

Abstract

Micro-C maps chromatin contacts by digesting crosslinked chromatin with micrococcal nuclease (MNase) and ligating DNA ends that are held in proximity. This protocol describes library preparation from Drosophila melanogaster embryos, including embryo collection, sequential crosslinking, nuclei isolation, MNase digestion, end processing and biotin labeling, proximity ligation, removal of biotin from unligated ends, DNA purification, and sequencing library preparation. Biotinylated DNA is enriched on streptavidin beads, converted into indexed libraries, and analyzed by paired-end sequencing. The workflow is intended for studies of chromosome architecture and regulatory contacts during embryogenesis.

Keywords: Micro-C; Drosophila embryos; chromatin architecture; micrococcal nuclease; boundary elements

Background

Understanding how chromatin architecture regulates gene expression requires mapping interactions at the scale of individual regulatory elements. Studies of genome architecture have been constrained by the resolution of traditional Hi-C, which relies on restriction enzyme digestion and produce DNA fragments spanning >5kb on average (Slobodyanyuk et al., 2022). Most regulatory elements span only a few hundred base pairs, and some enhancer–promoter pairs are separated by less than 5 kb. Consequently, Hi-C maps at resolutions coarser than 5 kb may fail to resolve many fine-scale regulatory interactions and small TAD structures.

Micro-C uses micrococcal nuclease (MNase) to fragment chromatin at a finer scale than conventional restriction enzyme-based chromosome conformation capture assays. MNase preferentially cleaves linker DNA between nucleosomes, generating fragments that allow chromatin contacts to be examined at nucleosomal resolution (<200 bp). Crosslinking preserves chromatin associations during digestion and end processing, while biotin enrichment isolates labeled DNA for sequencing. The resulting contact maps can reveal local chromatin folding and interactions within and between chromosomal domains (Slobodyanyuk et al., 2022).

In Drosophila embryos, Micro-C has been used to examine contacts involving the eve domain and its flanking boundaries, homie and nhomie (Bing et al., 2024; Ke et al., 2024; Fujioka et al., 2026). Functional dissection of homie provides complementary evidence for its roles in long-range regulation and insulation (Fujioka et al., 2025). Here, we present a detailed protocol used for Micro-C experiments in these studies.

Materials and reagents

Biological materials

Drosophila melanogaster adults of the required genotype, maintained in population cages for embryo collection on yeasted collection plates.

Reagents and commercial kits

Other common reagents

sodium hypochlorite solution; n-heptane; phosphate-buffered saline (PBS); Triton X-100; NP-40; Tween 20; dimethyl sulfoxide (DMSO); Tris base; HCl; NaCl; MgCl₂·6H₂O; CaCl₂·2H₂O; EDTA; EGTA; ATP; DTT; dTTP; dGTP; SDS; TE buffer; phenol:chloroform, pH 8.0; sodium acetate; absolute ethanol; tRNA, 2.5 mg/mL; nuclease-free water; and liquid nitrogen.

Use 10× NEBuffer 2.1, 10× NEBuffer 1, and 10× T4 DNA ligase buffer containing ATP for the reactions specified below. Prepare the working buffers in the Solutions and recipes section.

Laboratory supplies

Embryo collection cages and plates; brushes; glass vials; glass transfer pipettes; low-binding 1.5 mL microcentrifuge tubes; PCR tubes; Phase Lock Gel tubes or Phase Lock Gel; Covaris microTUBE-130 tubes (520045); and filtered pipette tips.

Solutions and recipes

Equipment

Use a population cage and a microscope for embryo staging; a horizontal or orbital shaker; a glass homogenizer and pestle; a refrigerated microcentrifuge and a mini centrifuge; a temperature-controlled thermomixer; a 65 °C water bath; a magnetic rack compatible with both microcentrifuge and PCR tubes; a thermal cycler; a vacuum concentrator; a Qubit fluorometer; a Bioanalyzer; a Covaris ME220 with the microTUBE-130 rack and matching acoustic waveguide; and an Illumina paired-end sequencer.

Software and datasets

Procedure

Critical Use consistent embryo input, developmental stage, and processing conditions across samples. Record the packed embryo volume, genotype, collection interval, incubation temperature, and biological replicate identifier.

A Embryo collection

A1.    Set up a large embryo collection cage using approximately four bottles of flies per cage. Allow the flies to acclimate for 1–2 days before collecting embryos.

A2.    For late-stage embryos, collect for 4 h, then incubate for a further 12–14 h at room temperature.

A3.    For nuclear cycle 14 (nc14) embryos, collect for 1 h, incubate for a further 2 h, and assess developmental stage by microscopy after fixation.

B Embryo fixation

Caution Handle formaldehyde, n-heptane, DMSO crosslinker stocks, and phenol:chloroform using the appropriate chemical hood and institutional protective measures.

B1.    Wash embryos from the collection plates and dechorionate with sodium hypochlorite solution for approximately 2–5 min, until the embryos float. Rinse away the bleach with water.

B2.    Add 5 mL water to a glass vial and transfer the dechorionated embryos into the vial with a brush. Add 7.5 mL n-heptane and mix well.

B3.    Using a glass pipette, remove the lower aqueous layer and material that has settled at the bottom, including debris, larvae, and yeast. Retain the embryos.

B4.    Add 5 mL PBST and 1.5 mL freshly opened 16% formaldehyde. Mix and fix for 15 min at room temperature on a horizontal shaker.

B5.    Vortex for 40 s to disperse the embryos. Allow the phases to separate for 2 min, then carefully remove as much of the upper phase as possible without losing embryos at the interface.

B6.    Add 10 mL PBST and vortex for 20 s. Allow the embryos to settle for 2 min. Transfer the settled embryos with a glass pipette to a 1.5 mL tube on ice.

B7.    Pellet the embryos briefly in a mini centrifuge and remove the supernatant with a glass pipette. Wash twice with 1 mL PBST, then remove as much wash buffer as possible.

Pause point Keep embryos on ice until all batches are ready. Embryos may be held on ice or at 4 °C for up to 24 h before the secondary crosslinking step.

B8.    Prepare the secondary crosslinking solution immediately before use, as described in Solutions and recipes. Add 1 mL to each embryo sample and incubate for 45 min at room temperature on a horizontal shaker.

B9.    Add 300 µL 2 M Tris-HCl, pH 7.5, mix, and incubate for 5 min at room temperature to quench the secondary crosslinking reaction.

B10.  Pellet the embryos briefly, discard the supernatant, and wash three times with 1 mL PBST. Allow the embryos to settle, then measure and record the packed embryo volume.

B11.     Remove as much PBST as possible with a P200 pipette, snap-freeze the embryos in liquid nitrogen, and store at −80 °C.

C Nuclei isolation and MNase digestion

C1.   Thaw TE–NP-40 and keep it on ice. Chill MB2 on ice. Add 1 mL TE–NP-40 to the fixed embryos and transfer the suspension to a glass homogenizer.

C2.   Homogenize with more than 20 pestle strokes, avoiding bubbles. Transfer the homogenate to a low-binding 1.5 mL tube.

C3.   Centrifuge at 10,000 × g for 1 min and discard the supernatant. Wash the nuclei pellet twice with 500 µL TE–NP-40, centrifuging at 10,000 × g for 1 min after each wash.

C4.   Remove the supernatant and resuspend the pellet in 500 µL TE–NP-40. Add the MNase while keeping the sample on ice; the working condition for Drosophila embryos is 4 µL of the 20 U/µL stock.

C5.   Mix for 1 h at 4 °C and 1,500 rpm.

C6.   On ice, add 3.5 µL 1 M MgCl₂ and 0.5 µL 1 M CaCl₂. Incubate for 45 min at 16 °C and 1,500 rpm.

C7.   Transfer the samples to ice and set the thermomixer to 65 °C. Add 15 µL 0.5 M EGTA to stop digestion, then incubate for 10 min at 65 °C and 1,000 rpm.

C8.   Centrifuge for 1 min at 10,000 rpm and discard the supernatant. Wash twice with 500 µL cold MB2, centrifuging for 1 min at 10,000 rpm after each wash. Remove the supernatant and set the thermomixer to 37 °C.

D End processing and biotin labeling

Prepare each mixture on ice for an approximately 25 µL nuclei pellet per reaction.

End-processing mixture per sample:

D1.   Add the end-processing mixture to the washed nuclei pellet and incubate for 20 min at 37 °C and 1,000 rpm. Centrifuge briefly to collect the liquid.

D2.   Add 2.5 µL Klenow fragment, 5 U/µL, and incubate for 15 min at 37 °C and 1,000 rpm. Prepare the end-labeling mixture during this incubation.

End-labeling mixture per sample:

D3.   Add the end-labeling mixture to the existing end-processing reaction. Incubate for 45 min at 25 °C and 1,500 rpm.

D4.   Transfer the samples to ice and set the thermomixer to 65 °C. Add 15 µL 0.5 M EDTA and incubate for 20 min at 65 °C.

D5.   Centrifuge for 1 min at 10,000 rpm and discard the supernatant. Wash once with 1 mL Micro-C wash buffer, then twice with 500 µL MB3. Centrifuge for 1 min at 10,000 rpm after each wash and remove the supernatant.

E Proximity ligation

Ligation mixture per sample:

E1.    Add the ligation mixture to the washed nuclei pellet. Incubate for 30 min at 25 °C and 1,000 rpm, then overnight at 16 °C and 400 rpm.

E2.    Centrifuge for 1 min at 10,000 rpm and remove the supernatant with a P200 pipette.

F Removal of biotin from unligated ends

Exonuclease III mixture per sample:

F1.    Add the exonuclease III mixture to the nuclei pellet and incubate for 15 min at 37 °C and 1,000 rpm.

G Reverse crosslinking

Reverse-crosslinking mixture per sample:

G1.   Add the reverse-crosslinking mixture to the exonuclease-treated sample and incubate overnight in a 65°C water bath.

Pause point After reverse crosslinking, samples may be stored at −20 °C before DNA purification.

H DNA purification

H1.   Remove the sample from the 65 °C water bath and centrifuge briefly for 30 s. Transfer the complete sample to a Phase Lock Gel tube, or add Phase Lock Gel to the sample tube.

H2.   Add 250 µL phenol:chloroform, pH 8.0, and vortex for 10 s. Centrifuge at maximum speed for 15 min, then transfer the upper aqueous phase to a new tube.

H3.   Add 25 µL 3M sodium acetate and 650 µL absolute ethanol. Mix well and precipitate for more than 1 h at −80 °C or overnight at −20 °C.

Pause point The precipitation mixture may be held at −20 °C before pelleting the DNA.

H4.   Centrifuge for 15 min at 4 °C. Remove the supernatant and wash the DNA pellet twice with 1 mL 70% ethanol. Remove the ethanol and dry the pellet in a vacuum concentrator.

H5.   Dissolve the DNA in 120 µL TE buffer.

I DNA shearing with the Covaris ME220

I1.     Transfer the DNA sample to a Covaris microTUBE-130 (520045). Power on the computer and ME220, allow at least 10 s for initialization, open Sonolab, and home the instrument.

I2.     Select the microTUBE-130 method and use the following settings.

I3.     Confirm that the instrument is homed. Load the acoustic waveguide that matches the sample rack, insert the rack and tubes, close the door, and start the method.

I4.     After the run, home the instrument and remove the samples, rack, and waveguide. Follow the instrument shutdown procedure, including draining the water bath and drying the treatment area.

I5.     Transfer the sheared DNA to a new tube and measure its concentration with the Qubit assay.

Pause point Store the purified, sheared DNA at −20 °C until streptavidin enrichment.

J Streptavidin enrichment

J1.    Wash 10 µL Dynabeads MyOne Streptavidin C1 with 1 mL TBW. Place the tube on a magnetic rack for 30 s and remove the supernatant. Repeat twice, for three washes in total.

J2.    Resuspend the beads in 110 µL 2× BW and add 6 µL tRNA, 2.5 mg/mL. Add the sheared DNA and incubate for 30 min at 25 °C and 1,000 rpm.

J3.    Place the tube on the magnetic rack for 30 s and remove the supernatant. Add 900 µL TBW and 5 µL tRNA, 2.5 mg/mL. Incubate for 3 min at 55 °C and 1,500 rpm, then centrifuge at 500 × g for 3 s.

J4.    Capture the beads on the magnetic rack and discard the supernatant. Wash twice more with 900 µL TBW.

J5.    Capture the beads for 30 s and remove the supernatant. Wash twice with 500 µL EB, retaining the beads on the magnetic rack. Remove the final supernatant and keep the beads on ice.

K Library end preparation and adapter ligation

K1.    Prepare 30 µL end-preparation mixture using 3.5 µL NEBNext Ultra II End Prep Reaction Buffer, 1.5 µL End Prep Enzyme Mix, and 25 µL water. Add to the beads and mix well.

K2.    Incubate for 30 min at 20 °C and 1,200 rpm, then for 30 min at 65 °C. Transfer the sample to ice.

K3.    Add, in order, 15 µL Ultra II Ligation Master Mix, 0.5 µL Ligation Enhancer, and 1.5 µL adapter. Mix well; the nominal liquid volume is 47 µL before USER treatment.

K4.    Incubate for 1 h at 20 °C and 1,200 rpm. Add 1.5 µL USER enzyme, mix, and incubate for 30 min at 37 °C and 1,200 rpm.

K5.    Add 200 µL TBW and incubate for 10 min at 55 °C and 1,200 rpm. Centrifuge briefly and transfer the suspension to a PCR tube.

K6.    Capture the beads on a magnetic rack for 30 s, remove the supernatant, and wash twice with 150 µL TBW. Remove the supernatant, then wash twice with 150 µL EB, keeping the beads immobilized on the magnetic rack. Remove the final wash and resuspend the beads in 23 µL water.

L PCR amplification and library cleanup

Critical Use the minimum number of PCR cycles needed to obtain sufficient library complexity. A separate qPCR is recommended to estimate the cycle number.

PCR mixture per sample:

L1.    Record the i5 and i7 primer identifiers and index sequences for each sample. Mix the PCR gently but thoroughly, ensuring that no bead clumps remain.

L2.    Run the following thermal-cycling program.

L3.    Add 45 µL AMPure XP beads to the 50 µL PCR and incubate for 10 min at room temperature. Capture the beads on the magnetic rack for 2 min and discard the supernatant.

L4.    With the beads on the magnetic rack, add 150 µL 70% ethanol, wait 30 s, and remove the wash. Repeat once. Open the tube and air-dry the beads for 5 min.

L5.    Add 30 µL water and mix by pipetting approximately 10 times. Incubate for 2 min at room temperature, capture the beads on the magnetic rack, and transfer the eluate to a new tube.

L6.    Measure library concentration with the Qubit assay. For a 200 µL assay, combine 199 µL prepared Qubit working solution with 1 µL sample. Analyze library size using the Bioanalyzer.

L7.    Sequence the libraries on an Illumina platform using a paired-end 100 bp sequencing kit.

Data analysis

First, align reads with BWA-MEM using -S -P -5 -M and an appropriate BDGP Release 6-based genome (dm6). Include verified engineered sequences when analyzing transgenic junctions. Use Pairtools to parse, sort, deduplicate, filter, and split alignments; exclude pairs with an unmapped mate or mapping quality below 3. Index pairs with Pairix, merge biological replicates after sample-level quality control, and generate 100 bp matrices and balanced multiresolution files with Cooler. For published virtual 4C analyses, FAN-C profiles were extracted at 400 bp resolution and smoothed over three bins. The analysis requires familiarity with Linux and contact-map file formats.

Use independent biological replicates for comparisons and retain separate sample-level quality-control reports. Predefine contact-map regions, normalization, and any inferential tests for the biological question. Report effect sizes and uncertainty across biological replicates rather than treating individual contacts or adjacent bins as independent biological observations.

Validation of protocol

This protocol builds on Micro-C methods described by Slobodyanyuk et al. (2022) and the Drosophila applications of Batut et al. (2022) and were used in Bing et al. (2024), Ke et al. (2024), Ke et al. (2026), and Fujioka et al. (2026). We report at least two independent biological replicates per genotype in these studies. Representative applications include the eve-domain analyses in Ke et al. (2024) and the transgene–eve contact maps in Figure 5 of Fujioka et al. (2026).

General notes and troubleshooting

MNase cleavage and recovery can vary with chromatin accessibility and fixation. A titration experiment to identify the right concentration is recommended.

Competing interests

The authors declare no competing interests.

Ethical considerations

The experimental material in this protocol is Drosophila melanogaster embryos. No ethical considerations is reported.

References

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3. Bing, X., Ke, W., Fujioka, M., Kurbidaeva, A., Levitt, S., Levine, M., Schedl, P. and Jaynes, J. B. (2024). Chromosome structure in Drosophila is determined by boundary pairing not loop extrusion. eLife 13:RP94070. https://doi.org/10.7554/eLife.94070

4. Fujioka, M., Ke, W., Schedl, P. and Jaynes, J. B. (2025). The homie insulator has sub-elements with different insulating and long-range pairing properties. Genetics 229(4):iyaf032. https://doi.org/10.1093/genetics/iyaf032

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