characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2016-03-01T17:02:04Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2016-02-11T15:45:16Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3870143
characteristics.characteristic: External Id
characteristics.text: University of Cambridge
characteristics.characteristic: INSDC center name
characteristics.text: 2016-03-01T17:02:04Z
characteristics.characteristic: INSDC first public
characteristics.text: 2016-02-11T15:45:16Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1057277
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-4462:Primed_HNES1_R1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: pluripotent stem cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0002248
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Human naive embryonic stem (HNES) cells were propagated in modified N2B27 medium supplemented with 1 uM PD0325901 (prepared in house), 1 uM CHIR99021 (prepared in house), 2.5 uM Go6983 (Sigma-Aldrich), 10 uM rho-associated kinase inhibitor (Y-27632, Calbiochem), 10 ng/ml human LIF (prepared in house) and 250 uM ascorbic acid (Sigma). N2B27 medium (1L) comprised 490 mL DMEM/F12 (Life Technologies), 490 mL Neurobasal (Life Technologies), 10 mL B27 (Life Technologies), 5 mL N2 (prepared in house), 10 ug/mL insulin (Sigma), 2 mM L-glutamine (Life Technologies) and 0.1 mM 2-mercaptoethanol (Sigma). N2 contains 100 ug/ml apo-transferrin (eBioscience, ABC2553), 3 uM sodium selenite (Sigma), 1.6 mg/mL putrescine (Sigma) and 2 ug/mL progesterone (Sigma) in DMEM/F-12 (Life Technologies). Cells were cultured in 5% O2, 7% CO2 in a humidified incubator at 37 C. Primed HNES cells were cultured in FGF/KSR medium, comprising 20% KnockOut Serum Replacement (Invitrogen), 1x non-essential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 uM 2-mercaptoethanol (Sigma), 10 ng/mL FGF2 (prepared in-house) in DMEM/F-12 basal medium (Sigma-Aldrich). Feeder cells were depleted by culture on gelatin-coated dishes for 40 minutes and harvesting cells in suspension. Pelleted cells were treated with lysis buffer (10 mM Tris-Cl pH 7.4 and 2% SDS) and 0.5 l proteinase K followed by incubation at 37 C for 1 h. Bisulfite conversion was performed on cell lysates. Whole-genome bisulfite libraries were prepared using a post-bisulfite adaptor tagging (PBAT) strategy as previously described (Smallwood et al. Nat Methods 11:817820) with some modifications. Bisulfite conversion was performed on Proteinase K digested cell lysates using the EZ DNA Methylation-Direct MagPrep Kit (Zymo). After purification, first-strand synthesis was performed using 6N-forward oligos for 37C for 30 min. Subsequently, samples were treated with Exonuclease I for 1 hour at 37 C, before DNA was purified using 0.8x Agencourt Ampure XP beads (Beckman Coulter). Samples were eluted in second-strand synthesis mix with 6N-reverse oligo and incubated at 37 C for 90 min. DNA was purified using 0.8x Agencourt Ampure XP beads and amplified with KAPA HiFi HotStart DNA Polymerase (KAPA Biosystems) using indexed iPCRTag primers (Quail et al. Nat Methods 9:10-11). Amplified libraries were pooled, purified using 0.8x Agencourt Ampure XP beads, and assessed for quality and quantity using High-Sensitivity DNA chips on the 2100 Bioanalyzer (Agilent) and the KAPA Library Quantification Kit for Illumina (KAPA Biosystems).
characteristics.characteristic: description
characteristics.text: embryo stage
characteristics.characteristic: developmental stage
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: embryo
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: Primed_HNES1_R1
characteristics.characteristic: title