title: Illumina Genome Analyzer IIx paired end sequencing; Hi-C in mouse fetal liver cells
design: Hi-C in mouse fetal liver cells
library_construction_protocol: Fetal livers were dissected from C57BL/6 mouse embryos at day 14.5 (E14.5) of development. Fetal liver cells were filtered through a cell strainer (70 μm) and directly fixed in formaldehyde. Hi-C was performed essentially as described in Lieberman-Aiden et al. (2009.), with some modifications. Thirty to 50 million cells were fixed in 2 % formaldehyde for 10 min, quenched with 0.125 M glycine, spun down (400 x g, 5 min) and washed once with PBS. The cells were incubated in 50 ml permeabilisation buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2 % Igepal CA- 630, Complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice with occasional agitation, spun down (650 x g, 5 min, 4 °C), and the cell pellets were resuspended in 358 μl of 1.25 x NEBuffer2 (NEB) per 5 million cell aliquot. Eleven μl of 10 % SDS was added to each aliquot, followed by an incubation at 37 °C for 60 min with continuous agitation (950 rpm). To quench the SDS, 75 μl of 10 % Triton X-100 was then added per aliquot, followed by an incubation at 37 °C for 60 min with continuous agitation (950 rpm). To digest chromatin, 1500 U of Hind III (NEB) was added per aliquot and incubated at 37 °C overnight with continuous agitation (950 rpm). After digestion, restriction sites were filled in with Klenow (NEB) in the presence of biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all 30 μM) for 60 min at 37 °C. For in-solution ligation, 86 μl of 10 % SDS was added per aliquot and incubated at 65 °C for 30 min with continuous agitation (950 rpm), followed by addition of 7.61 ml of ligation mix (745 μl of 10 % Triton X-100, 820 μl of 10 x T4 DNA ligase reaction buffer [NEB], 82 μl of 10 mg/ml BSA [NEB] and 5.965 ml water) per aliquot and incubation at 37 °C for 60 min with occasional agitation. For in-nucleus ligation, 7.61 ml of ligation mix (820 μl of 10 x T4 DNA ligase reaction buffer [NEB], 82 μl of 10 mg/ml BSA [NEB] and 6.71 ml water) was added per aliquot (compared to the in-solution ligation, SDS addition and incubation at 65 °C were omitted). For the ligation reaction (both in-solution and in-nucleus variants), 50 μl of 1 U/μl T4 DNA ligase (Life Technologies) was added per aliquot, followed by incubation at 16 °C for 4 h. The cross-links were reversed by adding 60 μl of 10 mg/ml proteinase K (Roche) per aliquot and incubating at 65 °C overnight. After overnight incubation, another 60 μl of proteinase K per aliquot was added, followed by incubation at 65 °C for an additional two hours. RNA was removed by adding 12.5 μl of 10 mg/ml RNase A (Roche) per aliquot and incubating at 37 °C for 60 min.  DNA was isolated by a phenol (Sigma) extraction, followed by a phenol/chloroform/isoamylalcohol (Sigma) extraction and standard ethanol precipitation. The precipitated DNA was washed three times with 70 % ethanol, and dissolved in 25 μl TE per aliquot. Subsequently all aliquots were pooled and the Hi-C DNA was quantified (Quant-iT Pico Green, Life Technologies).   Biotin was removed from non-ligated restriction fragment ends by incubating 30 to 40 μg of Hi-C library DNA with T4 DNA polymerase (NEB) for 4 h at 20 °C in the presence of dATP. After DNA purification (QIAquick PCR purification kit [Qiagen]) and sonication (Covaris E220), the sonicated DNA was end-repaired with T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB) and dNTPs in 1 x T4 DNA ligase reaction buffer (NEB). Double size selection of DNA was performed using AMPure XP beads (Beckman Coulter), before dATP-addition with Klenow exo- (NEB). Biotin- marked ligation products were isolated with MyOne Streptavidin C1 Dynabeads (Life Technologies) in binding buffer (5 mM Tris pH8, 0.5 mM EDTA, 1 M NaCl) for 30 min at room temperature, followed by two washes in binding buffer, and one wash in 1 x T4 DNA ligase reaction buffer (NEB). PE adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 h at room temperature (T4 DNA ligase in 1 x T4 DNA ligase reaction buffer [NEB], 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 NEBuffer 2. Bead-bound Hi-C DNA was amplified with 12 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) and quantitative PCR.
attributes.tag: Experimental Factor: fetal liver cell
attributes.value: cell type