GSM2154896: Mouse Hi-C; Mus musculus; OTHER
Cell pellets were incubated in 50 ml ice-cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal CA-630, protease inhibitor cocktail (Roche)) for 30 minutes on ice, followed by a centrifugation to pellet the cell nuclei (650 x g for 5 minutes). Nuclei were resuspended in 1.25 x NEBuffer 2, SDS was added (0.3% final concentration) and the nuclei were incubated at 37˚C with agitation (950 rpm) for one hour. Triton X-100 was added at a final concentration of 1.7 % and the nuclei were incubated at 37˚C with agitation (950 rpm) for one hour. Restriction digest was performed overnight at 37˚C with agitation (950 rpm) with HindIII (NEB; 1500 units per 5 million cells). The restriction sites were then filled in with Klenow (NEB) using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all at a final concentration of 30 µM) for 60 minutes at 37˚C. After addition of SDS (1.42 % final concentration) and incubation at 65˚C with agitation (950 rpm) for 25 minutes, ligation was performed for four hours at 16˚C (50 units T4 DNA ligase (Life Technologies) per 5 million cells starting material) in a total volume of 8.2 ml ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 µg/ml BSA, 0.9 % Triton X-100) per 5 million cells starting material. Reverse crosslinking (65˚C overnight in the presence of Proteinase K (Roche)) was followed by RNase A (Roche) treatment and two sequential phenol/chloroform extractions. The DNA was precipitated in the presence of sodium acetate 3 M pH 5.2 (1/10 volume) and ethanol (2.5 x volumes) overnight at -20˚C. After three consecutive washes with 70% ethanol, the DNA concentration was determined using Quant-iT Pico Green (Life Technologies). 40 µg of Hi-C library DNA were incubated with T4 DNA polymerase (NEB) for 4 hours at 20˚C to remove of biotin from non-ligated fragment ends, followed by phenol/chloroform purification and DNA precipitation overnight at -20˚C, followed by a 70% ethanol wash. Sonication was performed to obtain DNA fragments with a size peak around 400 bp (Covaris E220 settings: duty factor: 10%; peak incident power: 140W; cycles per burst: 200; time: 55 seconds). The sonicated DNA was end-repaired (T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB)) for 30 minutes at room temperature, followed by DNA purification (Qiagen PCR purification kit). dATP was added with Klenow exo- (NEB) for 30 minutes at 37˚C, and the enzyme was heat-inactivated (20 minutes at 65˚C). A double size selection using AMPure XP beads (Beckman Coulter) was performed: first, the ratio of AMPure XP beads solution volume to DNA sample volume was adjusted to 0.6:1. After incubation for 10 minutes at room temperature, the sample was transferred to a magnetic separator (DynaMag-2 magnet; Life Technologies), and the supernatant was transferred to a new eppendorf tube. The ratio of AMPure XP beads solution volume to DNA sample volume was then adjusted to 0.9:1 final. After incubation for 10 minutes at room temperature, the sample was transferred to a magnet (DynaMag-2 magnet; Life Technologies). Following two washes with 70% ethanol, the DNA was eluted in 100 µl of TLE (10 mM Tris-HCl pH 8.0; 0.1 mM EDTA). Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies) on a DynaMag-2 magnet (Life Technologies) in binding buffer (5 mM Tris pH8, 0.5 mM EDTA, 1 M NaCl) for 30 minutes at room temperature, followed by two washes in binding buffer, and one wash in ligation buffer (NEB). PE adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 hours at room temperature (T4 DNA ligase NEB, in ligation buffer, slowly rotating). After washes in wash buffer (5 mM Tris, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) and binding buffer, the DNA-bound beads were resuspended in a final volume of 30 µl NEBuffer 2. Bead-bound Hi-C DNA was amplified with 7 PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). The concentration and size distribution of Hi-C library DNA after PCR amplification was determined by Bioanalyzer profiles (Agilent Technologies). Hi-C libraries were paired-end sequenced (HiSeq 1000, Illumina).    To capture ligation products containing promoter sequences, 500 ng of Hi-C library DNA was lyophilized using a vacuum concentrator (Savant SPD 2010) at 45˚C. The DNA pellet was resuspended in 3.4 µl H2O, and custom hybridization blockers (Agilent Technologies) were added to the Hi-C DNA. Hybridization buffers and the custom-made RNA capture bait system (Agilent Technologies: biotinylated RNAs targeting the HindIII restriction fragment ends of 22,225 mouse gene promoters, and of 21,841 human gene promoters, respectively) were prepared according to the manufacturer’s instructions (SureSelect Target Enrichment, Agilent Technologies). The Hi-C library DNA was mixed with hybridization buffer, denatured for 5 minutes at 95˚C, and then incubated with the capture bait system at 65˚C. After 24 hours, biotin pulldown (MyOne Streptavidin T1 Dynabeads; Life Technologies) and washes were performed following the SureSelect Target enrichment protocol (Agilent Technologies). After the final wash, the beads were resuspended in 30 µl NEBuffer 2, without prior DNA elution. A post-capture PCR (four amplification cycles using Illumina PE PCR 1.0 and PE PCR 2.0 primers) was performed and the Capture Hi-C libraries were paired-end sequenced (HiSeq 1000, Illumina).
GEO Accession
GSM2154896