characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-11-08T17:03:32Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-12T11:29:36Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104679618
characteristics.characteristic: External Id
characteristics.text: EMBL Heidelberg
characteristics.characteristic: INSDC center name
characteristics.text: 2018-11-08T17:03:32Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-12T11:29:36Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS2281001
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6591:ES1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: 46C
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005484
characteristics.characteristic: cell line
characteristics.text: mouse embryonic stem cell
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0004038
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Sox1-GFP mouse embryonic stem cells derived from the E14tg2a line (46C, (Ying et al., 2003)) were used. Sox1-GFP mouse embryonic stem cells derived from the E14tg2a line (46C, (Ying et al., 2003)) were grown at 37 °C in a 5% (v/v) CO2 incubator in GMEM (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma), 2 ng/ml leukemia inhibitory factor (LIF, in-house), 1 mM 2-mercaptoethanol, non-essential amino acids (Gibco), L-glutamine (Gibco) and Na-pyruvate (Gibco), on gelatin-coated (0.1% v/v) plates. Accutase (Sigma) was used for cell dissociation. Cells were passaged every second day and seeded at a density of 1.3 million cells per 10 cm in surface area of the corresponding culture vessel. Medium was exchanged daily Libraries were produced following the in-situ HiC protocol published in Rao et al., 2014. In brief, cells were grown till 85% confluency and detached using Accutase. Cells were washed once in culture medium and once in PBS. 25M cell pellet was re-suspended in 50 ml medium and formaldehyde was added to a final concentration of 1%. Cells were incubated at room temperature with occasional mixing. The, 2.5ml of 2.5M Glycine was added and the reaction incubated for additional 5 minutes at room temperature. Cells were washed 2 times with ice cold PBS. Pellets were stored at -80. On the day of the experiment, 5M cell pellet were thawed for 10 minutes on ice. Cells were resuspended in 200 µl of lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630)) supplemented with 50µl of protease inhibitors (Sigma, P8340) for 15 minutes on ice. Nuclei were spun and washed once in lysis buffer. Nuclei were resuspended in 50 µl of 0.5% SDS and incubated at 62°C for 5 minutes. Sammples were next chilled to room temperature, 145μl of water and 25μl of 10% Triton X-100 was added and nuclei were incubated for 15 minutes at 37°C. Next, 25μl of 10X NEBuffer2 and 100U of MboI restriction enzyme (NEB, R0147) was added and chromatin digested overnight at 37°C with rotation. MboI was inactivated at 62°C for 20 minutes. DNA ends were labelled with biotinylated dATP. To this end 30 µl of the following mix was added to the sample: 37.5μl of 0.4mM biotin-14-dATP (Life Technologies, 19524-016), 1.5μl of 10mM dCTP, 1.5μl of 10mM dGTP, 1.5μl of 10mM dTTP and 8μl of 5U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210). Samples were incubated for 1.5 hours at 37°C with rotation. Then 900µl of ligation mix was added: 663μl of water, 120μl of 10X NEB T4 DNA ligase buffer (NEB, B0202), 100μl of 10% Triton X-100, 12μl of 10mg/ml Bovine Serum Albumin, 5μl of 400 U/ μl T4 DNA Ligase (NEB, M0202). Sampleswere incubated at room temperature for 4 hours with rotation. 50µl of 20mg/ml proteinase K 1and 20μl of 10% SDS were next added and the sample incubated for at 55°C for 30 minutes. Next, 130μl of 5M sodium chloride was added and the sample incubated at 68°C overnight. DNA was precipitated with 2 volumes of ethanol and washed 2 times with 75% ethanol. Pellet was air-dried and re-suspended in 100 µl of TE (10mM Tris, pH=8, 1mM EDTA). DNA was sheared to an average size of 400 bp using Covaris S2 apparatus. To size select fragments between 300 and 500 bp, 200 µl of DNA solution was mixed with 110μl (0.55X volumes) of AMPure XP beads (Beckman Coulter, A63881). Mix was incubated at room temperature for 5 minutes. Beads were separated on a magnet. The clear solution transferred to a fresh tube, 30μl of AMPure XP beads were added to the solution and incubated at room temperature for 5 minutes. Supernatant was discarded and the beads washed twice with 70% ethanol. DNA was eluted with 150 µl of 10 mM tris, pH=8. Biotinylated DNA was pulled down using streptavidin T1 beads (150 µl per reaction, washed once in Tween Wash Buffer: (1X TWB: 5mM Tris-HCl (pH 7.5); 0.5mM EDTA; 1M NaCl; 0.05% Tween 20) and re-suspended in 150µl of 2x Binding buffer (2X BB: 10mM Tris-HCl (pH 7.5); 1mM EDTA; 2M NaCl). Sample was incubated for 30 minutes at room temperature with mixing. After the incubation, beads were washed twice by incubating at 55°C for 2 min with 600 µl of 1xTWB (at each wash, the beads were separated from the wash buffer on magnet and the buffer discarded). Beads were washed once in 1x DNA ligase buffer. After this wash, beads were incubated at room temperature for 30 minutes with the blunting mix: 88μl of 1X NEB T4 DNA ligase buffer with 10mM ATP, 2μl of 25mM dNTP mix, 5μl of 10U/μl NEB T4 PNK (NEB, M0201), 4μl of 3U/μl NEB T4 DNA polymerase I (NEB, M0203), 1μl of 5U/μl NEB DNA polymerase I, Large (Klenow) Fragment (NEB, M0210). Beads were washed twice in TWB as indicated above and once in 100 µl 1x NEB buffer 2. Next neads were incubated at 37°C for 30 minutes with 100μl of dATP attachment master mix (90μl of 1X NEBuffer 2, 5μl of 10mM dATP, 5μl of 5U/μl NEB Klenow exo minus (NEB, M0212)). Beads were washed twice with 1x TWB (as above) and once with 100 µl of 1x quick ligation buffer (NEB M2200). Beads were re-suspended in 50 µl of quick ligation buffer and mixed with 3μl of an Illumina indexed adapter and 2 µl of quick ligase. After a 15 minute incubation, beads were washed twice with 1x TWB as above. Beads were re-suspended in 50 µl of 10 mM This, pH=8. Five amplification reactions were set, in each 5 µl of beads were mixed with 4 µl of 12.5 µM illumina primers and 25 µl of 2xphusion master mix and 16 µl of water. Samples were amplified for 6 cycles (according to manufacturer’s recommendations). DNA was purified using AmpureXP beads (60µl per 50 µl PCR reaction).
characteristics.characteristic: description
characteristics.text: Sox1-GFP
characteristics.characteristic: genotype
characteristics.text: 2i_LIF
characteristics.characteristic: growth condition
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: mouse embryonic stem cell
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0004038
characteristics.characteristic: progenitor cell type
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: ES1
characteristics.characteristic: title