GSM6589001: Hi-C LacZ Osmostress rep1; Drosophila melanogaster; Hi-C
After respective treatments, cells were harvested and crosslinked with 1% formaldehyde for 10 min followed by 5 min glycine treatment to quench crosslinking reaction, flash freezed and stored at 80°C. These cells were used for HiC library preparation. Cross-linked cells were incubated for 30 min on ice in HiC lysis buffer (10 mM Tris-HCl at pH 8, 10 mM NaCl, 0.2% NP-40), centrifuged for 5 min at 3500g, resuspended in 190 μL of NEBuffer2 1× (New England BioLabs [NEB]) and 10 μL of 10% SDS, and were then incubated for 10 min at 65°C. After the addition of Triton X-100 and 15-min incubation at 37°C, nuclei were centrifuged for 5 min at 3500g and were resuspended in 300 μL of 1x NEBuffer2. Nuclei were digested overnight using 400 U of the MboI restriction enzyme (NEB). For fill-in with bio-dATP, nuclei were pelleted and resuspended in fresh 1× repair buffer (1.5 μL of 10 mM dCTP, 1.5 μL of 10 mM dGTP, 1.5 μL of 10 mM dTTP, 37.5 μL of 0.4 mM biotin-dATP, 50 U of DNA Polymerase I large [Klenow] fragment in 300 μL NEBunffer2 1×). Ligation was performed for 4 h at 16°C using 10,000 units of T4 DNA ligase (NEB) in 1.2 mL of ligation buffer (120 μL of 10× T4 DNA ligase buffer, 100 μL of 10% Triton X-100, 12 μL of 10 mg/mL BSA, 963 μL of H2O). After reversion of the cross-link, DNA was purified using phenol/chloroform/isoamyl. Purified DNA was sonicated to an average size of 300–400 bp using a Bioruptor (Diagenode; eight cycles of 20 sec on and 60 sec off), and 3 μg of sonicated DNA was used per in situ Hi-C library preparation. Briefly, biotinylated DNA was pulled down using 20 μL of Dynabeads MyOne T1 streptavidin beads in binding buffer (5 mMTris-HCl at pH 7.5, 0.5 mM EDTA, 1 M NaCl). End-repair, A-tailing and Illumina adaptors ligation were performed on the beads using NEBNext library preparation end-repair and A-tailing modules (NEB). Libraries were amplified and sequenced in biological replicates.