MAGE-TAB Version 1.1 Investigation Title DNA methylation profiling of human naive embryonic stem cells Experimental Design growth condition design organism part comparison design cell type comparison design Experimental Design Term Source REF EFO EFO EFO Experimental Design Term Accession Number EFO_0001759 EFO_0001750 EFO_0001745 Experimental Factor Name cell type Experimental Factor Type cell type Experimental Factor Term Source REF EFO Experimental Factor Term Accession Number EFO_0000324 Person Last Name Bertone Person First Name Paul Person Mid Initials Person Email bertone@stemcells.cam.ac.uk Person Phone Person Fax Person Affiliation University of Cambridge Person Address Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2015-03-15 Public Release Date 2016-03-01 PubMed ID 26947977 Publication DOI 10.1016/j.stemcr.2016.02.005 Publication Author List Ge Guo, Ferdinand von Meyenn, Fatima Santos, Yaoyao Chen, Wolf Reik, Paul Bertone, Austin Smith, Jennifer Nichols Publication Title Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass Publication Status Publication Status Term Source REF Publication Status Term Accession Number Experiment Description Human pluripotent cell lines were derived from blastocyst-stage embryos and propagated in self-renewal conditions that maintain features of naive pluripotency characteristic of mouse embryonic stem cells. Genome-wide DNA methylation status of HNES1 and HNES3 naive and primed cells was assessed with post-bisulfite adapter tagging (PBAT). Protocol Name P-MTAB-48635 P-MTAB-48634 P-MTAB-48636 P-MTAB-48631 P-MTAB-48633 P-MTAB-48632 Protocol Type high throughput sequence alignment protocol nucleic acid sequencing protocol normalization data transformation protocol growth protocol nucleic acid library construction protocol nucleic acid extraction protocol Protocol Term Source REF EFO EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0004917 EFO_0004170 EFO_0003816 EFO_0003789 EFO_0004184 EFO_0002944 Protocol Description Sequencing reads were trimmed to remove the first 6 bases, adapter sequences and poor-quality reads using Trim Galore v0.3.8 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore, parameters: --clip_r1 6). The remaining sequences were mapped to human genome build GRCh37/hg19 using Bismark v0.13.1 (parameters: --bowtie2 --pbat) (Krueger and Andrews, Bioinformatics 27:1571-1572). Pooled libraries were prepared for 150bp single-end sequencing on the Illumina MiSeq or 125 paired-end sequencing on the Illumina HiSeq 2000. CpG methylation calls were extracted and analysed using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk) and custom R scripts. 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. 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). 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. Protocol Parameters Protocol Hardware Illumina HiSeq 2500 Protocol Software Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-4462.sdrf.txt Comment [Submitted Name] DNA methylation profiling of human naive embryonic stem cells Comment [SecondaryAccession] ERP014223 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR1255853-ERR1255864 Comment [AEExperimentType] methylation profiling by high throughput sequencing Comment[ArrayExpressAccession] E-MTAB-4462