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| Status |
Public on Sep 25, 2017 |
| Title |
P20 32F rRSeT methylC-seq |
| Sample type |
SRA |
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| Source name |
human induced pluripotent stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: naive-like hiPS cells derived from adult human dermal fibroblasts
age: 32
Sex: Female
identifier: 1569390
media: RSeT
reprogramming strategy: Sendai virus
passage number: 20 passages
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| Growth protocol |
to generate human primed and naïve iPSCs used for this study, somatic cell reprogramming with the Cytotune 2.0 kit, experiments were performed according to the manufacturer’s instructions (Invitrogen). Briefly, HDFa were seeded at ~5x10^4 cells per well of a 6-well plate in mouse embryonic fibroblasts (MEF) medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% nonessential amino acids (Gibco), 1mM GlutaMAX (Gibco), Pen-strep (Gibco), 0.1mM 2-mercaptoethanol (Gibco) and 1mM sodium pyruvate (Gibco) . After 2 days, cells were transduced with Sendai viruses in MEF medium with multiplicity of infections (MOIs) as follows, KOS MOI=5, c-MYC MOI=5, KLF4 MOI=6. After 24 hours, the medium was replaced with fresh MEF medium and media were changed every other day thereafter. On day 7, cells were trypsinized and seeded onto a layer of iMEF feeders or alternatively onto VTN-N (Gibco) coated T25 flasks at ~0.5x10^5~1x10^5 per flask in MEF medium. The next day, the cells were switched to different culture media. 21 days after transduction, cells were passaged and expanded using methods and media described below. Essential 8 medium: Add 10ml of Essential 8 supplement (Gibco) to 500ml medium basal (Gibco), supplement with 1% Pen-strep (Gibco); NHSM medium:DMEM/F12 medium (Gibco) (For 500ml) supplemented with 10mg/ml AlbuMAXI (Gibco), 1% Pen-strep (Gibco), 1mM GlutaMAX (Gibco), 1% nonessential amino acids (Gibco), 10% KnockOut Serum Replacement (Gibco), 5ml N2 supplement (Gibco), 12.5µg/ml recombinant human insulin (Sigma), 50µg/ml L-ascorbic acid (Sigma), 20ng/ml of recombinant human LIF (made in house), 8ng/ml FGF2 (Peprotech), 2ng/ml recombinant TGF-β1 (Peprotech), 20ng/ml human LR3-IGF1 (Prospec), and small molecule inhibitors: 1µM PD0325901 (Miltenyi Biotec), 3µM CHIR99021 (Miltenyi Biotec), 5µM SP600125 (Tocris), 2µM BIRB796 (Axon), 0.4µM LDN193189 (Axon), 10µM Y-27632 (supplemented daily to media from freshly thawed stock aliquot; abcam) and 1µM Gö6983 (supplemented daily to media from freshly thawed stock aliquot; Tocris); RSeT medium: Add 100ml of RSeT 5X Supplement, 1ml of RSeT 500X Supplement and 0.5ml of RSeT 1000X Supplement into 398.5ml of RSeT Basal Medium; (Stem cell technologies) supplement with 1% Pen-strep (Gibco); 5iLAF medium: A 50:50 mixture of DMEM/F-12 (Gibco) and Neurobasal medium (Gibco) supplemented with 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 1% nonessential amino acids (Gibco), 1mM GlutaMAX (Gibco), 1% Pen-strep (Gibco), 0.1mM 2-mercaptoethanol (Gibco), 50μg/ml BSA (Gibco), 1μM PD0325901 (Miltenyi Biotec), 1μM IM-12 (Millipore), 0.5μM SB590885 (Tocris), 1μM WH-4-023 (A Chemtek), 10μM Y-27632 (abcam), 20ng/ml Activin A (Peprotech), 8ng/ml FGF2 (Miltenyi Biotec), 20ng/ml human LIF (made in house) and 0.5% KSR (Gibco); t2iLGöY medium: A 50:50 mixture of DMEM/F-12 (Gibco) and Neurobasal medium (Gibco), supplemented with 2mM L-glutamine (Gibco), 0.1mM 2-mercaptoethanol (Gibco), 0.5% N2 supplement (Gibco), 1% B27 supplement (Gibco), 1% Pen-strep (Gibco), 10ng/ml human LIF (made in house), 250μM L-ascorbic acid (Sigma), 10µg/ml recombinant human insulin (Sigma), 1μM PD0325901 (Miltenyi Biotec), 1μM CHIR99021 (Miltenyi Biotec), 2.5μM Gö6983 (Tocris), 10μM Y-27632 (abcam). For conversion of established naive t2iLGöY cells to primed state, naive cells are trypsinized, counted and seeded onto VTN-N (Gibco) coated 6-well plates (in a range of density) in t2iLGöY media for 24 hours.The next day cells are transitioned to E8 medium (Gibco). Cells are then cultured and passaged as E8 primed cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNeasy Blood & Tissue Kit (Qiagen)
Following DNA extraction, 700ng-1µg of DNA for each sample was sheared to 200-bp using the Covaris DNA Shearing System. Methylated adapters (BIOO Scientific) were ligated to fragmented genomic DNA using the NxSeq AmpFREE Low DNA Library Kit (Lucigen). To capture regions of interest, we used baits provided in the SureSelectXT Human Methyl-Seq kit (Agilent) for enriching 84Mb covering 3.7 million CpGs. Sodium bisulfite conversion using the EZ-DNA Methylation Gold kit (Zymo Research) was performed on the enriched DNA followed by 11-14 cycles of polymerase chain reaction (PCR) amplification with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems). We performed sequencing of 101-bp single-end reads using the Illumina HiSeq 1500 sequencer as per the manufacturer’s instructions.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
Agilent SureSelect Methyl-Seq
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| Data processing |
Reads were quality and adapter trimmed with Cutadapt using the following parameters: -q 20,20 -m 30 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC. Trimmed reads were mapped to the in silico bisulfite converted human genome build hg19 using the bs-seeker2 software package (Guo et al, BMC Genomics, 2013, 14:774) with the following parameters: —aligner=bowtie —am=1. Methylation calling was also performed with bs-seeker2 using the default options. The heatmap of methylation levels in imprinted regions was made by calculating fraction of methyl-cytosine basecalls in the CpG context in each imprinted region and plotting the data with the pheatmap function in R.
Genome_build: hg19
Supplementary_files_format_and_content: allC - tab deliminted text files (chromosome, position, strand, context, methyl cytosine base calls, all base calls)
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| Submission date |
Aug 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ethan Edward Ford |
| E-mail(s) |
eford.dna@gmail.com
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| Phone |
0061 416 525 900
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| Organization name |
University of Western Australia
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| Department |
Plant Energy Biology
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| Lab |
Ryan Lister
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| Street address |
35 Stirling Highway
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| City |
Crawley |
| State/province |
WA |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE102851 |
Characterization of distinct states of human naive pluripotency |
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| Relations |
| BioSample |
SAMN07522701 |
| SRA |
SRX3110417 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2747178_P20_32F_Rset_R.allC.txt.gz |
202.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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