title: hESC culture
organism: Homo sapiens
description: Protocols: See Hrvatin et al. 10.1073/pnas.1400709111. Cells were washed twice with 2.5 ml/plate of ice-cold PBS and pelleted (800g, 5 minutes, 4C). The supernatant was aspirated and cells were resuspended 2.5 mL/plate Buffer A containing 15 mM Tris HCl (pH 8), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (Ambion, pH 8), 0.5 mM EGTA (pH 8) and 0.5 mM Spermidine (Sigma-Aldrich). An equal volume of 2X Igepal in Buffer A (earlier samples with a 0.05% Igepal final concentration, later samples with a 0.00165% Igepal final concentration) was added to the resuspended cells and the nuclei were pelleted (800g, 5 minutes, 4C). The supernatant was aspirated promptly and the cells resuspended in 2.5 mL/plate Buffer A. An aliquot of the resuspended nuclei was taken and counted on a hematocytometer with 1:10 Trypan Blue. Nuclei were pelleted (800g, 5 minutes, 4C) and either digested immediately as below, or stored in 20 mM Tris HCl (pH 8), 75 mM NaCl, 0.5 mM EDTA (Ambion) 50% glycerol, 0.85 mM DTT, and 0.125 mM PMSF, flash frozen in liquid nitrogen and stored at -80C for a later digestion. If frozen, nuclei were washed twice with 2.5 mL/plate Buffer A, pelleted (800g, 5 minutes, 4C) and the supernatant discarded before proceeding to digestion. Digestion volumes are given for 10e7 nuclei. Volumes should be scaled as per cell number. Digestion buffer (850 ul per 107 nuclei) was prepared by diluting 10X stock of CaCl2 and NaCl in Buffer A (above) for a final concentration of 6 mM CaCl2 and 75 mM NaCl. Stop buffer (950 ul per 10e7 nuclei) was prepared with final concentrations as follows: 50 mM Tris HCl (pH 8), 100 mM Na Cl, 0.1 % SDS, 100 mM EDTA (Ambion, pH 8), 10 ug/ml RNase A (Invitrogen), 1 mM Spermidine (Sigma-Aldrich) and 0.5 M Spermine (Sigma-Aldrich). Both digestion and stop buffers were warmed at 37C. DNaseI enzyme was combined with warmed digestion buffer for a final volume of 100 ul per 107 nuclei. If multiple concentrations of DNaseI were tested, nuclei were separated equally amongst each concentration. Prior to digestion, a small aliquot was reserved as an undigested control for quantitative PCR analysis. The DNaseI/ digestion buffer mix was warmed in a 37C water bath for 2 minutes, and the nuclei were gently resuspended in digestion buffer. The warmed DNaseI/ digestion buffer mix was promptly added to the nuclei and digested for 2 minutes at 37C. Pre- warmed stop buffer was added and the tube inverted multiple times to stop the digestion. Digested samples were transferred to a 55C incubator for 15 minutes. Proteinase K (Ambion, 20 mg/mL) was added to each sample for a final concentration of 2ug/mL and samples were incubated overnight at 55C. DNA fragments were purified by adding an equal volume of phenol:chloroform:isoamyl alcohol to each sample and mixing. The suspension was transferred to a phase-lock tube and centrifuged for 5 minutes at 12-16,000g. The aqueous phase was isolated and combined with 0.1 volumes 3M sodium acetate, 2 volumes absolute ethanol and 1:600 Glycoblue. This mixture was incubated at -20C for 5-6 hours or at -80C for 30-45 minutes. DNA was pelleted by centrifugation at 12-16000g for 10-20 minutes and the supernatant aspirated. The DNA pellet was washed with 70% ethanol and re-pelleted by centrifugation at 4000 rpm for 5 minutes. The supernatant was carefully aspirated and the DNA pellet air-dried for 5-10 minutes before resuspending in TE buffer. To enrich for hypersensitive regions, size selection was performed on the purified fragments by either 2% agarose gel band purification or the Invitrogen E-Gel Agarose System. Fragments were collected in the 175-400 bp region so as to avoid empirically-determined complications by the 150 bp nucleosomal signal. Size selection enrichment was tested by quantitative PCR using primers (three positive control primers and three negative control primers) for constitutively DNase hypersensitive regions and insensitive regions as positive and negative controls respectively, on both the samples as well as the reserved undigested controls. Size selection enrichment was calculated as follows: Enrichment = 2^((Ct(negative control primer)-Ct(positive control primers))digested, size selected fragments-(Ct(negative control primer)-Ct(positive control primers))undigested control). NA
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-03-24T17:04:22Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T23:57:17Z
attributes.tag: External Id
attributes.value: SAMEA3311865
attributes.tag: INSDC center name
attributes.value: Massachusetts Institute of Technology
attributes.tag: INSDC first public
attributes.value: 2015-03-24T17:04:22Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T23:57:17Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3432:hESC culture
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: Hues8
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-3432:hESC culture
attributes.tag: scientific_name
attributes.value: Homo sapiens