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| Status |
Public on Dec 13, 2014 |
| Title |
RRBS of primed WIBR3 ESCs |
| Sample type |
SRA |
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| Source name |
Primed WIBR3 ESCs
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| Organism |
Homo sapiens |
| Characteristics |
cell line: WIBR3
cell type: hESC
pluripotent state: Primed
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| Growth protocol |
Primed Cells were expanded in DMEM/F12, 20%KSR and 8ng/ml FGF2 on irradiated Feeder cells. Rock inhibitor was added 2-24 hours before splitting with trypsin. Naïve cells were expanded in WIS-NHSM conditions that include: KO-DMEM (Invitrogen 10829-018, 03-049) 500ml, Pen-strep 5ml (Biological Industries 03-033-1B), L-glutamine 5ml (Biological Industries 02-022-1B), NEAA – 5ml (Biological Industries 01-340-1B), 5gr Albumax I (Invitrogen - 11020021) (dissolve in media bottle for 30 minutes in RT)
- Insulin (Sigma I-1882) - add 6.25mg insulin per 1 bottle to give approximately additional 12.5microg/ml insulin)
- Apo-transferrin (Sigma T-1147), 100 μg/ml final concentration
- Progesterone (Sigma P8783), 0.02 μg/ml final concentration;
- Putrescine (SigmaP5780), 16 μg/ml final concentration
- Sodium selenite (Sigma S5261), add 5 μl of 3 mM stock solution per 500ml of medium.
- L-ascorbic acid 2-phosphate (Sigma A8960) (50 μg/ml final concentration)
- 1) human LIF (in house produced or Peprotech # 300-05) – 10 μg total (~20ng/ml final concentration) (2 vials)
- 2) FGF2 (Peprotech 100-18c or Rnd systems –4114-TC-01M) 8ng/ml final (1 vial)
- 3) TGFB1 – 1ng/ml final (Peprotech 100-21c-1000) (0.5 vial)
- 4) Chir99021 (Axon 1386)– 3μM final (1/3 vial)
- 5) PD0325901 (Axon 1408)– 1μM final (1 vial)
- 6) p38i SB203580– 5μM final(1 vial)
- 7) JNKi SP600125 (TOCRIS 1496)– 5μM final (1 vial).
- 8) ROCKi Y27632 (Axon 1683)-10μM final (2 vials or add fresh 1:1000 every media change)
- 9) PKCi (Go6983 – TOCRIS 2285)- 0.5μM (1 vial or add 1:2000 fresh every media change)
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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 |
WIBR3_primed
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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 hg19 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: hg19
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 associated publication. 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 12, 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 |
GPL16791 |
| 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 |
SAMN03263892 |
| SRA |
SRX807558 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1564930_WIBR3_primed_new.cpgs.txt.gz |
18.7 Mb |
(ftp)(http) |
TXT |
| GSM1564930_WIBR3_primed_orig.cpgs.txt.gz |
18.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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