|
| Status |
Public on Jun 23, 2016 |
| Title |
WT_RAS_eRRBS |
| Sample type |
SRA |
| |
|
| Source name |
Primary HSPC Cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Lin- enriched hematopoietic stem/progenitor cells
genotype: DNMT3A WT with NRAS G12D
time point: day 16 post-transduction
|
| Treatment protocol |
Primary HSPC Cells were retrovirally infected with NRAS G12D alone (EV-RAS), DNMT3A R882H with NRAS G12D (RH-RAS) or DNMT3A WT with NRAS G12D (WT-RAS)
|
| Growth protocol |
Lin- enriched murine hematopoietic stem/progenitor cells leukemia progenitor cell lines were cultured in Opti-MEM base medium supplemented with 10% FBS, 1% antibiotics, 50 μM mercaptoethanol and stem cell factor
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen).
eRRBS was carried out with a previously described protocol with slight modification (Garrett-Bakelman et al., 2015). Briefly, ~300 ng of genomic DNA were digested with three different enzymes (~80 units of MspI, 40 units of BfaI and 40 units of MseI) to enhance genomic fragmentation and coverage. The produced fragments were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine, followed by overhang fill-in, 3’-terminal-A extension and purification. Bisulfite treatment of the fragments was done using the EZ DNA Methylation–Lightning kit (Zymo Research). Amplified eRRBS libraries was quality checked with Agilent 2200 TapeStation, followed by deep sequencing on the Illumina HiSeq-2000 genome analyzer with 50 bp SE parameters.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Sequence reads from bisulfite-treated libraries were identified using standard Illumina base-calling software.
Residual cytosines (Cs) in each read were first converted to thymines (Ts), with each such conversion noted for subsequent analysis. A reference sequence database was constructed from the 50-bp ends of each computationally predicted MspI-TaqI fragment in the 40–350 bp size range.
All Cs in each fragment end were then converted to Ts; the converted reads were aligned to the converted reference by Bowtie. The number of mismatches in the induced alignment was then counted between the unconverted read and reference, ignoring cases in which a T in the unconverted read is matched to a C in the unconverted reference. For a given read, the best alignment was kept. If there was more than one best alignment, the read was discarded as non-unique.
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.
Genome_build: mm9
Supplementary_files_format_and_content: bigBed files contain all mapped CpGs with the number of methylated reads and number of total reads listed.
|
| |
|
| Submission date |
Jul 29, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Rui Lu |
| E-mail(s) |
[email protected]
|
| Organization name |
Univ of Alabama at Birmingham
|
| Street address |
1824 6th Ave S
|
| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE71473 |
Effect of DNMT3A R882H mutation or WT expression on global DNA methylation patterns of hematopoietic stem/progenitor cells with NRAS G12D co-transduction (eRRBS) |
| GSE71475 |
Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene expression program and acute leukemia development |
|
| Relations |
| BioSample |
SAMN03941724 |
| SRA |
SRX1122922 |
