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| Status |
Public on Sep 10, 2014 |
| Title |
H9_Primed3 |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
condition: Conventional
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| Treatment protocol |
To induce resetting, transfected PSC expressing NANOG and KLF2 were dissociated with trypsin and replated as single cells in the presence of ROCK inhibitor. Doxycycline (DOX, 1μM) was added the next day and 24h later medium was changed to N2B27 medium (Ndiff227, StemCells Inc., SCS-SF- NB-02) with 1μM PD0325901 (PD03), human LIF (prepared in-house), DOX and either 3μM CHIR99021 (2iL+DOX) or 1μM CHIR99021 (t2iL+DOX). Medium was changed every day. Cells were split every five to seven days after dissociation to single cells with Accutase (Life Technologies) for 10 minutes. Around two weeks after DOX induction, cells were transferred to t2iL+5μM PKC inhibitor Gö6983 (Sigma-Aldrich) (t2iL+Gö). Reset cells cultured in t2iL+Gö were also passaged every five to seven days as single cells. Cells transferred to t2iL+Gö proliferate slowly for the initial couple of passages after withdrawal of DOX. Cells were maintained on MEF feeders throughout.
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| Growth protocol |
Human embryo derived cells, H9 (WiCell Research Institute) (Thomson et al., 1998) were cultured on mouse embryonic fibroblast (MEF) cells. Conventional PSC cell medium (FGF/KSR) comprised DMEM/F12 (Sigma-Aldrich) with 10ng/ml bFGF (prepared in-house) and 20% KSR (Invitrogen) supplemented with 100 mM 2-mercaptoethanol (Sigma-Aldrich, cat. M7522), MEM nonessential amino acids (Invitrogen, cat. 11140050), 2 mM L-glutamine (Invitrogen, cat. 25030024). Conventional PSC were passaged every five to seven days as small clumps by dissociation with 0.025% Trypsin, 1mg/ml Collagenase IV (Invitrogen17104-019), KSR (final 20%), 1mM CaCl2. Throughout this study cells were maintained in incubators at 5% oxygen.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared using AllPrep DNA/RNA mini kit (QIAGEN)
Genomic DNA was fragmented by sonication (Covaris) and adaptor ligated using Illumina supplied methylated adaptors and NEBnext library preparation kit. Subsequently DNA was bisulfite-treated using the Sigma Imprint kit according to the manufacturer’s instructions (one step protocol). Final library amplification (11 cycles) was done using KAPA Uracil+ (KAPA Biosystems), after which the libraries were purified using Ampure beads (x1).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
Raw sequence reads were trimmed to remove both poor quality calls and adapters using Trim Galore (www.bioinformatics.babraham.ac.uk/projects/trim_galore/) (v0.3.3, default parameters).
Remaining sequences were mapped to the human GRCh37 genome using Bismark (Krueger and Andrews, 2011) (v0.9.0, parameters --bowtie2); CpG methylation calls were extracted and analysed using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk/) and custom R scripts. Global methylation comparison was calculated by averaging 1kb window methylation levels of CpGs covered by at least 30 reads.
Genome_build: GRCh37
The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
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| Submission date |
Aug 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL15433 |
| Series (1) |
| GSE60945 |
Resetting Transcription Factor Control Circuitry Towards Ground State Pluripotency In Human |
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| Relations |
| BioSample |
SAMN03013627 |
| SRA |
SRX690033 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1493985_H9_Primed3.cov.txt.gz |
208.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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