48hpf_fibroblast_2
Protocols: 1. We followed the general protocol for in situ Hi-C as described in (Rao et al., 2014) which we adapted for use on a lower cell numbers. Mainly, differences were related to adjustments on the volume of the reactions, a shortening of the digestion step, a removal of biotin from the un-ligated fragments, and an alternative strategy for size-selection during library preparation. Embryos were reared following Westerfield et al., (2009) recommendations. Briefly, once collected on a Petri dish filled with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 and 0.00001% (w/v) of Methylene blue) at 28°C, 10 embryos per replicate were manually dechorionated using a pair of Dumont's #5 forceps. Dechorionated embryos were rinsed thrice with 400 μL of warmed Ringer's solution (116 mM NaCl, 2.9 mM KCl, 5.0 mM HEPES at pH7.2) and collected into a 1.5 mL eppendorf tube using a plastic Pasteur pipette. Then the tube was placed on ice to sedate the embryos. The tube containing the embryos was then removed from the ice, the liquid was fully removed and the embryos were resuspended in 200 μL of Deyolking buffer (Ringer's solution, 0.3 mM PMSF, 1 mM EDTA). Energetic up and down movements with a 200 μL pipette were done in order to separate yolks from embryos. Once the clean embryos sunk down the liquid was fully removed and the embryos were rinsed twice with Ringer's solution and embryos were finally resuspended in 400 μL PBS and homogenized using a syringe (size 24G). 2. Cross-linking. In a fume hood, cells were cross-linked on a 1% final concentration (v/v) of 37% Formaldehyde and vigorously shaken prior to 10 min room temperature incubation with gentle rotation (20 rpm). The reaction was quenched for 5 min at room temperature and with gentle rotation (20 rpm) by adding 2.5M Glycine solution to a final concentration of 0.2M. Cells were pelleted twice (300 g, 4°C for 5 min) and resuspended on 1 ml of cold 1X PBS. 4. DNA precipitation. DNA was precipitated by adding 630 μL Phenol-Chloroform-Isoamyl alcohol (25:24:1) and samples were spun (5 min at 17,000 g). The supernatant was then mixed to 63 μL of 3M sodium acetate pH5.2, 2 μL GlycoBlue (Life Technologies) and 1008 μL pure ethanol, followed by 15 min incubation at -80°C. Samples were then spun at 17,000 g at least for 30 min. The supernatant was discarded and the pellet was washed with cold 70% ethanol. All ethanol traces were removed, the pellet was air-dried for no longer than 5 min, dissolved in 30 μL of Tris pH 8.0 (Applichem) and incubated for 5 min at 37°C without rotation. RNA was digested by adding 1 μL RNAse to each sample and the mix was incubated 15 min at 37°C. Biotin from unligated fragments was removed by adding 10 μL of 10X NEB2 buffer (New England Biolabs), 1mM of a dNTPs mix (2.5 μL), 20mg/ml BSA (0.5 μL), nuclease-free water (up to 100 μL) and 3U/μL T4 DNA polymerase (5 μL; New England Biolabs). DNA shearing. Samples were mixed by gentle pipetting up and down and incubated at 20°C for 4h with no rotation. Samples were brought to a 120 μL volume with nuclease-free water and DNA was then sheared using a Covaris S220 instrument (2 cycles, each 50 sec long; 10% duty; 4 intensity; 200 cycles/burst). Biotin Pull-down. Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) were used to pull down biotinylated fragments according to manufacturer's guidelines (https://tools.thermofisher.com/content/sfs/manuals/dynabeads_myone_savC1_man.pdf). Briefly, 120 μL of the sheared Hi-C sample was mixed with an equal amount of the prepared magnetic beads and incubated for 15 min with rotation to allow DNA binding to the beads. The beads were washed twice with 1x B&W buffer + 0.1% Triton X-100 by incubating the samples at 55°C with shaking (1000 rpm) for 2 min, followed by a wash with 10 mM Tris-Cl pH 8.0 and finally resuspended in 50 μL of the last wash solution. 3. Lysis. Cells were pelleted and gently resuspended in 500 μL of ice-cold in situ Hi-C lysis buffer (10 mM Tris-Cl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, cOmplete Ultra protease inhibitors). Following 15 min incubation on ice, cells were spun down (1000 g, 4°C for 5 min) and pellet was resuspended in 250 μL of ice-cold in situ Hi-C lysis buffer. Lysed cells were then flash-frozen in liquid Nitrogen and stored at -80°C. Restriction Enzyme digestion. Frozen aliquots at different cell concentrations were placed on ice to thaw, spun (13,000 g for 5 min at 4°C) and resuspended in 500 μL lysis buffer. Following yet another spun (13,000 g for 5 min at 4°C), cells were gently resuspended in 500 μL ice-cold 10x NEB2 buffer. Nuclei were permeabilized by resuspending them in 50 μL of 0.4% SDS and incubating for 10 min at 65°C without agitation. The SDS was then quenched by adding 25 μL of 10% Triton X-100 and 145 μL of nuclease-free water, respectively, and incubating at 37°C for 45 min with rotation (650 rpm). Chromatin was digested by adding 100U of MboI in 25 μL of 10x NEB2.1 buffer, respectively (New England Biolabs). All the digestions were performed at 37°C with gentle rotation for a period of 90 min by adding the restriction enzyme in two installments. After the digestion, MboI samples were heat-inactivated for 20 min at 62°C. Marking of DNA Ends. The overhangs generated by the restriction enzyme cuts were filled-in by adding a mix of 0.4 mM biotin-14-dATP (18.75 μL; Life Technologies), 10mM dCTP, 10mM dGTP and 10mM dTTP (0.75 μL of each dinucleotide), and 5U/μL DNA polymerase I Klenow (New England Biolabs), followed by a 90 min incubation at 37°C with gentle rotation. Proximity ligation. The DNA fragments were then ligated by adding a master mix containing nuclease-free water (657 μL), 10X T4 DNA ligase buffer (120 μL), 10% Triton X-100 (100 μL), 20mg/ml BSA (12 μL) and 5 Weiss U/μL T4 DNA ligase (5 μL in two installments; Thermo Fisher Scientific). Samples were mixed by inversion and incubated at 20°C with gentle rotation for 4 h. Crosslink reversal. The crosslink was reverted by spinning down the nuclei (2500 g for 5 min at room temperature) and resuspending them in 500 μL extraction buffer. To digest the proteins 20 μL of 20mg/ml proteinase K (Applichem) was added to the mix, followed by 30 min incubation at 55°C with rotation (1000 rpm). After the incubation, 130 μL of 5M sodium chloride was added and incubated overnight at 65°C with shaking (1000rpm).
ENA first public
2022-01-19
ENA last update
2022-01-19
External Id
SAMEA8651410
INSDC center alias
Max Planck Institute for Molecular Biomedicine
INSDC center name
Max Planck Institute for Molecular Biomedicine
INSDC first public
2022-01-19T00:17:35Z
INSDC last update
2022-01-19T00:17:35Z
INSDC status
public
Submitter Id
E-MTAB-63131619541027:48hpf_fibroblast_2
broker name
ArrayExpress
common name
zebrafish
developmental stage
hatching long-pec
genotype
TL strain
restriction enzyme
MboI
sample name
E-MTAB-63131619541027:48hpf_fibroblast_2