Day -2 (-3): Prefixing procedures, blood-feeding
1. We highly recommend using the colony with 100-150 mature females for producing a sufficient amount of mosquito eggs. Blood-feed the adult mosquito with fresh blood (0-5 days old)1. Allow the females to develop ovaries based on a species-specific schedule.
Day 0: Egg laying
1. Put the 10 cm Petri dish with wet cotton balls covered with filter paper according to the biological schedule of mosquito strain for overnight egg-laying2. An example of the egg-laying dish is provided in Fig.1.
Day 1: Embryo collecting and bleaching
1. The next day total amount of eggs must be counted before starting the Hi-C experiment to make sure that there is a sufficient number of eggs for further manipulations. Due to the cell loss at the next protocol steps, we recommend using not less than 2000-4000 embryos for generating a qualitative Hi-C library.
2. After calculating, carefully collect eggs from the wet filter paper and incubate in presence of 50%-bleach-water solution for 10 minutes with shaking to unseal extraembryonic membranes (amnion and serosa)3.
3. Accurately monitor the time of incubation; depending on the structure of covers, which should be slightly “spotted” but not overdigested, duration of bleaching may vary but takes on average about 8-12 minutes4.
Fixation, cell lysis, and restriction
1. Freshly prepare and pre-chill all buffer solutions (Fixing buffer with PFA, Fixing buffer w/o PFA, Fixing buffer with glycine from Supplementary file 1) before you start the experiment.
2. Collect eggs after bleaching, remove the leftovers of bleach solution with filter paper, wash 2-3 times with ddH2O, remove the leftovers of any liquid, and add 10 mL fixing buffer with PFA (final concentration 3%).
3. Homogenize the collected eggs with Dounce’s or any alternative homogenizer for 2 minutes, filter with 100 µm nylon mesh (Millipore), collect cells, and leave for crosslinking for 15 minutes at RT with slow rotation.
4. Filter the cells with nylon mesh (~40-60 µm, Millipore) to decrease the income of cell aggregates, incubate for additional 10 minutes.
5. Quench PFA by adding an appropriate amount of fixing buffer with glycine (20 mL per 10 mL buffer with PFA, total glycine concentration 1M) and incubate on ice for 5 minutes.
6. Collect cells with centrifugation at 4⁰C, 3000 rpm, 15 minutes.
7. Wash with cold 20 mL fixing buffer w/o PFA twice (first, resuspend the pellet into homogenous single-cell suspension in 1 mL of buffer, then add the rest of buffer).
8. Wash again with pre-chilled 1.25XNeb 2.1 buffer (buffer specialized for restriction endonuclease of your choice) and reduce the reaction volume to 1-1.5 mL (avoid any abrupt temperature shifts).
9. Collect cells with centrifugation at 4⁰C, 4000rpm, 15 min.
10. After discarding the supernatant nuclei pellet should be quickly frozen in liquid nitrogen and stored at -80⁰C for up to several months or resuspended in 300 µL restriction buffer for continuing with Hi-C protocol (*checkpoint – K0, take an ⅕ aliquot for library verification).
11. Take frozen cells aliquot and carefully defrost it on ice for ~10-15 minutes, resuspend in ~300 µL restriction buffer.
12. To degrade DNA-unbound proteins add 10-20% SDS to a total concentration of 0.3% and incubate at 37°C for 1 hour with shaking, 1000 rpm.
13. Quench SDS by adding 1/5*V 10% triton (~70-80 µL) and incubate at 37°C for 1 hour with shaking, 1000 rpm.
14. After quenching add 150-200U restriction enzyme (MboI or DpnII), accurately mix the reaction by pipetting and incubate overnight (12-15 hours) at 37°C with slowly overhead rotation (for preventing cells sticking), 300-500 rpm.
Day 2: Biotin-14-dATP marking of DNA ends, ligation
1. The next day, once the restriction is completed, the reaction solution becomes clearer and more transparent. To inactivate MboI-directed restriction incubate reaction mix at 62°C for 25 minutes, 1000 rpm.
2. During inactivation progress, prepare Klenow mix to full-fill the sticky ends with biotin-dATP (Supplementary file 1).
3. Incubate for 45-90 minutes at 37°C with rotation, 500-1000 rpm.
4. While biotin-labeling is in progress, prepare ligation mix (Supplementary file 1). Keep T4-ligation buffer, BSA, T4-ligase, and ATP on ice before using.
5. Set up a 1 mL-pipette at 1000 µL point and accurately mix the ligation mix by pipetting before adding to the main reaction. Mix the ligation reaction thoroughly by pipetting and incubate at RT (20-25⁰C) with slow rotation, 300-500 rpm, overnight (15-18 hours).
6. In 2 hours add a fresh portion of T4-ligase and ATP to maximize ligation efficiency.
Day 3: Crosslink reversal
1. When ligation has been completed, add 7 µL 10 µg/µL RNase A to degrade any present amount of RNA, incubate for 1 hour at 37°C with mixing, 1000rpm.
2. To remove protein-DNA crosslinks add 50 µL proteinase K (20 µg/µL), incubate for 1 hour at 56°C with shaking, 1000 rpm.
3. Then, add 0.1V 5M NaCl (V – the volume of reaction), incubate at 68°C for at least 2 hours or overnight5.
Day 4 (or Day 3 - continued): DNA purification, Hi-C library check-in
1. After overnight incubation at 68°C cool the reaction to RT and split into two aliquots for more convenience. To eliminate the presence of any embryo covers or agglutinated cell conglomerates we suggest using classical phenol-chloroform-isoamyl* purification (or phenol/chloroform) with adding a drop of vacuum grease sterilized by autoclaving6, 7.
2. Add 1V phenol-chloroform-isoamyl solution, mix thoroughly, centrifuge at max speed for 15 minutes.
3. Carefully transfer the clear upper phase to the new 2 mL DNA LoBinding tube (lower phase and middle phase contain all other impurities such as proteins residues, eggs covers, excluding DNA).
4. Then, continue to perform ethanol DNA extraction according to suggested proportions: add 1.6V pure ethanol and 0.1V 3M NaOAc, incubate for 1-2 minutes at RT with rotation, transfer tubes at -80°C, and keep for 15 minutes (or until the liquid becomes frozen), centrifuge at 4°C at max speed for 15 minutes.
5. A visible white/light yellow pellet should precipitate at the tube bottom. Transfer tubes on ice and leave for a couple of minutes before discarding the supernatant. Carefully wash DNA pellet twice with pre-chilled 800 µL 70-80% ethanol, then centrifuge at RT, max speed.
6. Accurately mix the pellet each time avoiding cells sticking to the plastic tips and tubes possibly. The actual size of the pellet should become less after the second washing because of dissolving SDS, DTT, and ATP particles at RT.
7. After washing steps perform air-drying for 5 minutes, then dissolve the pellet with 130µL 10mM Tris-HCl, incubate at 37°C for 15 minutes for better efficiency, and split the solution into 10µL (for Qubit, 2µL+PCR 1µL+ 5µL gel electrophoresis) and XµL aliquots (for COVARIS, depending on instrument technical requirements). Keep samples at -20°C before the next steps.
8. Perform High Sensitive Qubit assay to estimate DNA concentration using the recommended protocol and check the library quality by PCR-analysis with specified 3C primers. 10-200ng/µL range (1.3-25µg in total) is optimal and sufficient quantity to continue with the Hi-C protocol.
Day 5 (or Day 4): DNA shearing + size selection
1. After verifying the DNA concentration of the Hi-C library, continue with ultrasonic DNA shearing. The optimal COVARIS requirements should be chosen based on BioAnalyzer/TapeStation or regular gel electrophoresis results given that the majority of DNA fragments have to be performed as a smear between 300-500 bp on electrophoregram.
2. Accurately transfer sheared DNA to the fresh tube, wash COVARIS tube with ddH2O, increase the reaction volume to exactly 200 µL, and perform 300-500 bp DNA fragments purification and size selection with Agencourt® AMPure XP beads following to recommended protocol.
3. Pre-warm AMPure XP beads to RT, mix thoroughly before using, add exactly 0.55X volumes (110 µL) to the reaction, mix by pipetting, and incubate at RT for 5 minutes.
4. Keep the liquid containing DNA fragments shorter than 500 bp by separating beads at the magnetic rack for 2-5 minutes. Transfer the clear solution to the new tube.
5. To purify 300-500 bp DNA fragments from RNA and short fragments add a new portion of AMPure beads - 0.15X (30 µL) – to the main reaction, mix thoroughly, and incubate for 5 minutes at RT. Separate beads at the magnetic rack, keep the beads.
6. Wash with freshly made 70%-ethanol two times without agitation, keep the beads on a magnet all the time. 7. Evaporate the small amount of ethanol by air-drying for 3-7 minutes, but be careful and avoid overdrying – beads should have dark brown color without any visible drops of ethanol, light color of pellet, and small cracks evidence for overdrying which can lead to decreasing in DNA yield.
8. Dissolve the pellet in 312 µL 10 mM Tris-HCl, TE buffer, or regular ddH2O. Estimate DNA concentration by dsDNA High Sensitivity Qubit Assay and run 10 µL of purified solution on 2% agarose gel to verify the size of selected DNA fragments. Keep the tube at -20°C or continue with the next step.
Day 6 (or Day 5): biotin pull-down + library preparation for Next Generation Illumina sequencing
1. We suggest aliquot the beads mix for single-use samples to prevent extra heating each time.
2. Pre-heat Dynabeads to RT. Thoroughly wash 100 µL of 10mg/µL Dynabeads with 400 µL 1 x Tween Washing Buffer (5mM Tris-HCl, 0.5mM EDTA, 1M NaCl, 0.05% Tween 20), separate on a magnetic rack.
3. Resuspend the beads in 300 µL 2 x Binding Buffer (10mM Tris-HCl, 1mM EDTA, 2M NaCl) and add to the main reaction.
4. Mix thoroughly and incubate at RT for 15 minutes with slow mixing to enhance binding between biotinylated DNA and streptavidin beads.
5. After incubation, separate Dynabeads on a magnet for 5 minutes.
6. Discard the supernatant, wash the beads twice with Tween Washing Buffer with 2-minutes incubation at 55°C each time, then wash with 150 µL 10mM Tris-HCl buffer without heating. Do not intensively mix the beads during the washing steps.
7. Separate the beads on a magnetic rack (discard supernatant, keep the beads).
8. After the last washing, add 50 µL 10 mM Tris-HCl buffer, resuspend the mixture thoroughly, transfer to the PCR tube, and continue with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® Index Primers Set 1 to amplify the library directly on streptavidin beads.
Adapter ligation with NEBNext® Ultra™ II DNA Library Prep Kit
For adapter ligation and Hi-C-library amplification follow the recommended instructions and index compatibility recommendations provided by NEB for NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with minor changes9. Here, we describe the procedure in detail to maximize the reproducibility of the results.
1. Defrost all the reagents on ice, mix thoroughly, and add NEBNext Ultra II End Prep Enzyme Mix, NEBNext Ultra II End Prep Reaction Buffer, and Hi-C library diluted in 10 mM Tris-HCl to the new nuclease-free PCR tube.
2. Mix the reaction by pipetting up and down 10-20 times without extensive bubbling, perform quick spin, but make sure that Dynabeads are mixed evenly and not laying at the bottom.
3. Place in the thermocycler with ≥75°C heat lid setting, incubate at 20⁰C for 15 minutes, mix the beads directly at thermocycler, and incubate at 20°C for 15 minutes more.
4. Thoroughly mix the beads avoiding bubbling, incubate at 65°C for 15 minutes, mix directly at thermocycler, and incubate for 15 minutes more. Hold at 4°C before proceeding with the next steps.
5. Defrost on ice NEBNext Ultra II Ligation Master Mix, NEBNext Ultra II Ligation Enhancer, NEBNext Ultra II Adapter for Illumina, add to the main reaction, and mix thoroughly 10-20 times by pipetting. Total reaction volume can differ because of the volume covered by beads but should be in the range ~95-100 µL in total.
6. Perform a quick spin to prevent any on-tube-side-left drops but then make sure that beads are spread across the entire reaction volume evenly. Incubate at 20°C (heated lid off) for 20 minutes with intermediate pipetting the beads.
7. Add 3 µL USER Enzyme, mix well, and incubate at 37°C for 15 minutes with the lid setting heated to ≥47⁰C. 8. After incubation, transfer the reaction mixture to DNA LoBinding 1.5 mL tube and perform cleanup from an extensive amount of adaptor. Discard the supernatant containing adaptors by separating the beads on a magnetic rack.
9. Wash twice with 600 µL Tween Washing Buffer with 2-minutes heating at 55⁰C, separate beads for at least 5 minutes.
10. Wash the beads with 100 µL 10 mM Tris-HCl, separate on a magnet, discard the liquid phase, and add 10 µL 10 mM Tris-HCl to perform PCR.
PCR library amplification
We would suggest splitting the total reaction volume into two aliquots and performing PCR for one-half of the Hi-C library to verify the optimal quantity of PCR cycles based on the final amount of DNA. Usually, 9-10 cycles are sufficient but in case of some DNA loss during the washing or pull-down steps, it may be necessary to increase the number of cycles to 11-12.
1. Defrost the reagents (NEBNext Ultra II Q5 Master Mix, Universal PCR Primer, Index primer), combine with Adaptor ligated Hi-C library on the Dynabeads, and transfer to the sterile PCR tube to a total volume of 25 µL.
2. Perform PCR according to the manufacturer's recommended conditions.
3. Mix the reaction directly into the thermocycler after the 3rd and 6th cycles to increase the efficiency of the reaction.
4. After PCR is completed, add 125 µL 10 mM Tris-HCl or ddH2O, separate the beads on a magnetic rack for 5 minutes, and keep the supernatant (discard the beads).
5. Transfer supernatant to the new DNA LoBind 1.5 mL tube and perform AMPure beads cleanup.
6. To select DNA fragments upper than 400 bp add exactly 0.7 volume of pre-heated to RT AMPure beads, pipette thoroughly, incubate for 5 minutes at RT,
7. Separate on a magnet, and keep the beads! Wash twice with 70-80% freshly diluted ethanol excluding any agitation.
8. Remove any drops of ethanol, perform air-drying for 3-5 minutes at RT until AMPure beads pellet becomes slightly light and wrinkled. Do not allow overdrying which can affect the solubility of DNA.
9. Dissolve Hi-C library in 30 µL 0.1 x TE buffer and measure DNA concentration by High Sensitivity dsDNA Qubit. If the total amount of DNA after PCR is sufficient for NGS-sequencing10, tubes should be stored at -20-80°C until sequencing. If the producing amount of DNA is not enough – perform PCR for another half increasing the number of cycles to 11-12. Verify the concentration by High Sensitivity Qubit dsDNA assay.