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| Status |
Public on Sep 18, 2020 |
| Title |
Tet/Tet neurons |
| Sample type |
SRA |
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| Source name |
Differentiated neurons derived from ESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: Differentiated neurons derived from ESCs
genotype: Dnmt1 Tet/Tet
cell line: Tet/Tet
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| Treatment protocol |
None
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| Growth protocol |
R1 and Tet/Tet ESCs were cultured in ES media composed of Knock out DMEM F12, supplemented with FBS, NEAA, Glutamax, β-Mercaptoethanol, Penicillin /Streptomycin and LIF. Tet/Tet cells were grown in presence of Puromycin (1ug/ml) and Neomycin (150ug/ml). On reaching confluency (i.e 1 × 106 cells), 1/6th of the cells were seeded for embryoid-body(EB) formation. The culture media is ES medium without LIF. After 2 days, these EBs are induced to form neurons for 10-15 days in Ndiff227 media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell pellets from trypsinized R1 and Tet/Tet ESCs were resuspended in 1X PBS and centrifuged at 1,000 rpm for five min at room temperature in a swing-out rotor. The pellets were washed once again with 1X PBS, resuspended in a lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 50 mM NaCl, 0.5% SDS) and treated with Proteinase K (50µg/ml) at 50℃ for two hours. The cell lysates were extracted with phenol: chloroform (1:1 ratio), centrifuged at 12,000 rpm for 10 min at room temperature and the supernatants were extracted with chloroform and centrifuged in the same manner. DNAs in the supernatants were precipitated using sodium acetate (final concentration = 0.3 M, pH 5.2) and an equal volume of isopropanol for five minutes by gentle inversions. The precipitated DNAs were spooled using sterile glass rods, washed with 70% ethanol, dried at room temperature for 15-30 minutes and dissolved in 1X TE buffer (10mM Tris-Cl, 1mM EDTA, pH 8.0). In case of neurons, confluent dishes of neurons were scraped with lysis buffer(RLT buffer with BME) and further QIAGEN All Prep Kit(Cat No. 80204) procedure was followed for the DNA extraction. The extracted DNAs were then quantified using NanoDrop.
200 ng of starting input genomic DNA was digested with 30 units of MspI (NEB) and then purified with Zymo Research DNA Clean & Concentrator™-5 (Cat#: D4003). Fragments were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine according to Illumina’s specified guidelines. Adaptor-ligated fragments ≥50 bp in size were recovered using the DNA Clean & Concentrator™-5 (Cat#: D4003). The fragments were then bisulfite-treated using the EZ DNA Methylation-Lightning™ Kit (Cat#: D5030). Preparative-scale PCR was performed and the resulting products were purified with DNA Clean & Concentrator™-5 (Cat#: D4003) for sequencing on an Illumina HiSeq.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
zr2544_2
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| Data processing |
Sequence reads from bisulfite-treated Classic RRBS libraries were identified using standard Illumina base calling software and then raw FASTQ files were adapter, filled-in nucleotides and quality trimmed using TrimGalore 0.6.4.
FastQC 0.11.8 was used to assess the effect of trimming and overall quality distributions of the data
Alignment to the mm10 reference genome was performed using Bismark 0.19.0.
Methylated and unmethylated read totals for each CpG site were called using MethylDackel 0.3.0. The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T.
Fisher’s exact test was performed for each CpG site that has at least five reads coverage, 10% difference in methylation value between groups and a p-value (unadjusted) of < 0.05. in order to identify significantly differentially methylated cytosines.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph files, UCSC browser tracks
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| Submission date |
Jun 19, 2020 |
| Last update date |
Sep 18, 2020 |
| Contact name |
Kommu Naga Mohan |
| E-mail(s) |
[email protected]
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| Phone |
4066303608
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| Organization name |
Birla Institute of Technology, Pilan- Hyderabad Campus
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| Department |
Biological Sciences
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| Lab |
Mohan Laboratory
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| Street address |
shameerpet, Jawaharnagar
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| City |
Hyderabad |
| State/province |
Telangana |
| ZIP/Postal code |
500078 |
| Country |
India |
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| Platform ID |
GPL18480 |
| Series (1) |
| GSE152817 |
Reduced Representational Bisulfite Sequencing of R1 and Dnmt1 Tet/Tet mouse embryonic stem cell lines |
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| Relations |
| BioSample |
SAMN15325254 |
| SRA |
SRX8582113 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4626849_Tet_neu_RRBS_meth_calling_new.txt.gz |
27.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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