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| Status |
Public on Dec 23, 2014 |
| Title |
RRBS of 129 EpiSCs reverted to a naïve state |
| Sample type |
SRA |
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| Source name |
129 EpiSCs
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| Organism |
Mus musculus |
| Characteristics |
cell line: 129
cell type: EpiSC
pluripotent state: Naïve
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| Growth protocol |
All cells were grown in N2B27-based media: 500ml KO-DMEM (Invitrogen), 5ml N2 supplement (Invitrogen), 5ml B27 supplement (Invitrogen), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM b-mercaptoethanol (Sigma), penicillin/streptomycin (Invitrogen), 5 mg/ml BSA (Sigma). Primed EpiSCs were grown in N2B27 with 8ng/ml recombinant human bFGF (Peprotech Asia), 20 ng/ml recombinant human Activin (Peprotech Asia) and 1% KSR. The EpiSCs were grown on feeder-free gelatin/vitronectin- or Matrigel-coated plates. Primed EpiSCs were reverted into a naive state by growing them 2i/LIF conditions: N2B27 with 5ug recombinant human LIF (Peprotech), 3mM CHIR99021 ( Axon Medchem) and 1mM PD0325901 (TOCRIS). Naive cells were grown on gelatin-coated plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from snap-frozen cell pellets (Quick-gDNA MiniPrep, Zymo Research). 300ng of DNA was then digested overnight with MspI (NEB R0106) followed by end-repair (NEB E6050) and addition of a 3’ overhang A using Klenow fragment (3'-5' Exo-, NEB M0212). The resulting DNA fragments were size-selected for ones shorter than 500bp using AMPure XP beads (Beckman Coulter A63881). 10ng of size-selected DNA was ligated, using quick T4 ligase (NEB M2200), into a cut and T-tailed plasmid containing a 6bp unique molecular identifier (UMI) sequence. The ligated plasmid was bisulfite converted and cleaned (EZ DNA methylation Gold kit, Zymo Research). The converted DNA was amplified using 25 cycles of PCR with GoTaq Hot Start polymerase (Promega M5005) and primers matching specific locations on the plasmid. The primer sites were cut from the amplicons using XbaI (NEB R0145) end EcoRI-HF (NEB R3101). Further details regarding the plasmid are available in Shipony Z et al. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells. Nature 2014 Sep 4;513(7516):115-9.
RRBS fragments underwent addition of a 3’ overhang A using Klenow fragment (3'-5' Exo-, NEB M0212). The fragments were then ligated to Illumina compatible adapters using quick T4 ligase (NEB M2200) and amplified using 8 cycles of PCR with GoTaq Hot Start polymerase (Promega M5005) and primers matching the Illumina adapters. The final products underwent gel electrophoresis using NuSieve 3:1 agarose (Lonza 50094). Gel bands matching fragment sizes of 200-500bp were cut and the DNA was extracted (MinElute Gel Extraction Kit, Qiagen 28604). Libraries were then pooled and sequenced on HiSeq 2500 system according to the manufacturer’s procedures.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
129_naive
RRBS
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| Data processing |
FASTQ files were generated using the Illumina CASAVA 1.8.2 software.
The plasmid introduces a CTAGANNNNNNATTGATT prefix at one read and an AATTCAATT prefix on the other read (were NNNNNN is the UMI). These prefixes were removed and the UMI recorded for each fragment. A small fraction of reads include longer prefixes that result from amplicons that did not undergo digestion by XbaI or EcoRI. These reads were identified and the longer prefixes were removed as well.
The reads were aligned to the mm9 reference genome using bismark version 0.7.7. Alignment was performed independently for each read of the read-pair. We expect each read to start at an MspI restriction site. Therefore we discarded reads that failed to align within 1bp of such sites. We also discarded read-pairs where each read aligned to remove MspI sites. On the other hand, in cases where one read uniquely mapped on a restriction site but its pair could not be mapped uniquely or could not be mapped at all, we attempted to re-align the entire read pair to the fragment.
To avoid PCR biases we grouped reads that map to the same MspI restriction site, have the same UMI and were ligated into the plasmid in the same orientation (positive or negative). Only the first such read was maintained and all other reads were dropped.
The methylation state of each CpG was extracted from the bismark output. For each CpG, the number of reads in which it was methylated or unmethylated was counted and recorded.
Genome_build: mm9
Supplementary_files_format_and_content: Tab-separated text files containing CpG methylation values. The files' columns are: Column1: chromosome. Column 2: position of CpG. Column 3: seen count. Column 4: methylation count. Note that each CpG's coverage is given by column 3, and its methylation value by column4/column3. Files ending with _orig.cpgs.txt represent the data used in the paper. Files ending with _new.cpgs.txt represent the output of a newer, improved version of our computational pipeline.
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| Submission date |
Dec 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Department |
Molecular Genetics
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| Street address |
Weizmann Institute
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| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE52617 |
Derivation of novel human ground state naïve pluripotent stem cells [ChIP-seq; RRBS-seq] |
| GSE52824 |
Derivation of novel human ground state naïve pluripotent stem cells. |
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| Relations |
| BioSample |
SAMN03272760 |
| SRA |
SRX823550 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1571968_129_naive_new.cpgs.txt.gz |
9.1 Mb |
(ftp)(http) |
TXT |
| GSM1571968_129_naive_orig.cpgs.txt.gz |
9.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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