GSM2811197: S2 cell rep3; Drosophila melanogaster; Hi-C
S2 cells were scraped, transferred to 50 mL Falcon tubes and the volume adjusted to 25 mL with Schneider medium. Formaldehyde was added to a final concentration of 2% and the cells were fixed for 10 min at 25°C. The fixation was quenched with 25 mL 2 M glycine and placing on ice. Cells were centifuged, washed in cold PBS, then counted by haemocytomety. 2x10^7 cell aliquots were resuspended in 5 mL nuclear permeabilisation buffer (15 mM HEPES pH 7.6, 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 0.1% Triton-X100, 0.5 mM DTT, protease inhibitor cocktail (Roche)) and left on ice for 30 min. Nuclei were centrifuged then resuspended in 300 µl 1.25 x Dpn II buffer (New England Biolabs) plus 0.3% SDS and incubated for 1 hr at 37°C, 1000 rpm on a shaker. Triton-X100 was added to a final concentration of 2% and the nuclei were incubated for a further 1 hr at 37°C, 1000 rpm, before overnight digestion with 1500 U Dpn II (New England Biolabs) at 37°C, 1000 rpm. The restriction enzyme was inactivated by incubation for 20 min at 65°C, 1000 rpm with SDS at a final concentration of 1.3%, before dilution of the chromatin in 10 ml 1x T4 DNA ligase buffer (New England Biolabs) plus 1% Triton-X100 and incubation for 1 hr at 37°C, 750 rpm. The chromatin was ligated for 4 hr at 25°C, 750 rpm with 40,000 U T4 DNA ligase (New England Biolabs) then crosslinks were reversed overnight at 65°C, 750 rpm in the presence of 150 µg/ml proteinase K. The 3C DNA was then purified by 1 hr treatment with 40 µg/ml RNase A at 37°C, 750 rpm, phenol extraction, phenol/chloroform extraction and ethanol precipitation. The DNA was quantified with the Qubit dsDNA assay (Invitrogen). Aliquots of 5 µg 3C DNA were sonicated in a Bioruptor (Diagenode) in 50 µl volumes in sonication buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS) to obtain a smear between 500-1500 bp. The sonicated 3C DNA was then purified by phenol/chloroform extraction and ethanol precipitation and quantified with the Qubit dsDNA assay (Invitrogen). Libraries for paired-end sequencing were made from 500 ng aliquots of sonicated 3C DNA using Illumina reagents and protocols, with size selection for products of ~800 bp.
GEO Accession
GSM2811197