title: GSM6761335: Hi-C, N.furzeri rep1; Nothobranchius furzeri; Hi-C
library_construction_protocol: The Hi-C procedure used to map 3D genomic interactions in killifish was adapted from previous studies as follows. Trypsinized killifish fibroblasts (2.5 X 10^6) were crosslinked using 37% formaldehyde (Sigma) at a final concentration of 1% for 15  min at room temperature. The formaldehyde was then quenched with 2.5 M glycine (at a final concentration of 0.25 M), and the cells were centrifuged at 2,000g for 5 min, washed once with 1× PBS, and stored at −80 °C. For use, the cells were lysed in 250 µl of cold Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl,0.2% Igepal CA630) and incubated on ice for 15 minutes, followed by centrifugation at 2,500 g for 5 minutes, a wash with 500 µL cold Hi-C Lysis Buffer, and centrifugation at 2,500 g for 5 minutes. The pellet was resuspended in 50 µl 0.5% SDS, and incubated at 62◦C for 10 minutes. The SDS was quenched by adding 145 µl H2O and 25 µl 10% Triton X-100, followed by a 15-minute incubation at 37◦C. Restriction digestion was carried out by adding 25 µl of 10× NEBuffer 2 and 100 U of the MboI restriction enzyme (NEB, R0147), followed by a 2 h incubation at 37◦C in a Thermomixer at 900 rpm. The reaction was then incubated at 62 °C for 20 min to inactivate the restriction enzyme. Fragment ends were filled in by adding: 37.5 µl 0.4 mM biotin-14-dATP (ThermoFisher Scientific, # 19524-016); 1.5 µl each of 10 mM dCTP, dGTP, and dTTP; and 8 µl of 5 U µl−1 DNA Polymerase I Large (Klenow) Fragment (NEB M0210). The reaction was incubated at 37 °C in a Thermomixer at 900 rpm. for 45 min. Fragment end ligation was carried out by adding 663  µl H2O, 120  µl 10× NEB T4 DNA ligase buffer (NEB B0202), 100  µl 10% Triton X-100, 12  µl BSA (10  mg/ml, NEB) and 5  µl 400  U/µl T4 DNA ligase (NEB M0202), and incubating at room temperature for 4  h with rotation. Nuclei were then pelleted by centrifugation at 3,500 g for 5 minutes, resuspended in 130 µl ChIP Elution Buffer (1% SDS, 0.1 M NaHCO3), supplemented with Proteinase K, and incubated at 65◦C overnight to reverse crosslinking. Samples were then sonicated using a Covaris E220, and the DNA was purified using the MinElute PCR Purification Kit (Qiagen #28006), with elution in a total volume of 300 µ1× EB buffer. Biotin-labeled DNA was purified on 20 µl of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602), which were separated on a magnetic stand, washed with 400 µl of 1× TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20), and resuspended in 300 µl of 2× Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl). The sonicated DNA was added to the beads and the mixture was incubated for 15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 600 µl 1× TWB, and were heated at 55◦C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55◦C incubation were repeated. Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows: End repair was carried out by resuspending the beads in 50 µl 1× EB buffer, and adding 3 µl NEB Ultra End Repair Enzyme and 7 µl NEB Ultra End Repair Enzyme, followed by incubation at 20◦C for 30 minutes, and then at 65◦C for 30 minutes. Adapters were ligated to DNA fragments by adding 30 µl Blunt Ligation mix, 1 µl Ligation Enhancer, and 2.5 µl NEB Adapter, incubating at 20◦C for 20 minutes, and then adding 3 µl USER enzyme, and incubating at 37◦C for 15 minutes. Beads were then separated on a magnetic stand and washed with 600 µl TWB for 2 minutes at 55◦C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, the beads were washed in 100 µl 0.1 × TE buffer, then were resuspended in 16 µL 0.1 × TE buffer, and heated at 98◦C for 10 minutes. For PCR, 5 µl of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 µl 2× NEB Ultra PCR Master Mix. The reaction was incubated at 98◦C for 30 s followed by 12 cycles of 98◦C for 10 s, 65◦C for 30 s, and 72◦C for 1 minute, followed by incubation at 72◦C for 5 minutes. The beads were separated on a magnetic stand, and the supernatant was cleaned with 1× AMPure XP beads. Libraries were sequenced in a paired-end format on an Illumina NextSeq instrument using NextSeq 500/550 high output kits.