title: GSM3820082: Cell26_scHiC; Drosophila melanogaster; Hi-C
library_construction_protocol: We used previously published protocol of single-nucleus Hi-C [PMID:28355183] with minor modifications. 5-10 million cells were fixed in 1× phosphate buffered solution (PBS) with 2% formaldehyde for 10 minutes with occasional mixing. The reaction was quenched with by addition of 2 M glycine to a final concentration of 125 mM. Cells were pelleted by centrifugation (1,000 g, 10 minutes, 4 °C), resuspended in 50 μl of 1× PBS, snap-frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 1.5 ml of isotonic buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% (v/v) NP-40 substitute (Fluka), 1% (v/v) Triton-X100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific)) on ice for 15 minutes. Cells were pelleted by centrifugation at 2,500g for 5 minutes, resuspended in 100 μl of 1× DpnII buffer (NEB) and pelleted again. The pellet was resuspended in 200 μl of 0.3% SDS in 1.1× DpnII buffer and incubated at 37 °C for 1 hour. Then 330 μl of 1.1× DpnII buffer and 53 μl of 20% Triton X-100 (Sigma) were added, and the suspension was incubated at 37 °C for 1 hour. Next, 600 U of DpnII enzyme (NEB) were added, and the chromatin was digested overnight (14-16 hours) at 37 °C with shaking (1,400 rpm). In the morning, 200 U of DpnII enzyme were added and the cells were incubated for additional 2 hours. DpnII was then inactivated by incubation at 65 °C for 20 minutes. Nuclei were pelleted by centrifugation at 3,000 g for 5 minutes, resuspended in 100 μl of 1× T4 DNA ligase buffer (Fermentas) and pelleted again. The pellet was resuspended in 400 μl of 1× T4 DNA ligase buffer, and 75 U of T4 DNA ligase (Fermentas) were added. Chromatin fragments were ligated at 16 °C for 6 hours. Next, the nuclei were pelleted by centrifugation at 5,000 g for 5 minutes, resuspended in 100 μl of sterile 1× PBS, stained with Hoechst, and single nuclei were isolated into wells of a standard 96-well PCR plate (Thermo Fisher) using FACS (BD FACSAriaTMIII). Each well contained 3 μl of sample buffer from illustra GenomiPhi v2 DNA amplification kit (GE Healthcare). Sample buffer drops containing isolated nuclei were covered by 5 μl of mineral oil (Thermo Fisher), incubated at 65 °C for 3 hours to reverse formaldehyde cross-links. Total DNA was amplified according to a previously published protocol [PMID:18533898]. The amplification was considered successful if the sample contained ≥1 μg DNA. Samples were prepared for sequencing using Illumina TruSeq reagents (NEB) according to the manufacturer's instruction.
attributes.tag: GEO Accession
attributes.value: GSM3820082