|
| Status |
Public on Jun 23, 2016 |
| Title |
HSPC_WTRAS_INPUT |
| Sample type |
SRA |
| |
|
| Source name |
Primary HSPC Cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype: DNMT3A WT with NRAS G12D
chip batch: batch 1
chip antibody: none
cell type: Lin- enriched hematopoietic stem/progenitor cells
|
| 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 |
Lysates were clarified from sonicated nuclei and DNMT3A R882H- or histone-DNA complexes were isolated with indicated antibodies.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
All reads were mapped to the mouse genome (mm9) using the BWA alignment software. Unique reads mapped to a single best-matching location with no more than two mismatches were kept, which were then subject to removal of reads generated by PCR- caused duplicates using the Picard and Samtools.
Profiles of ChIP-Seq read densities were displayed in the Integrative Genomics Viewer (IGV, Broad Institute) and the Integrative Genomics Browser (IGB, Affymetrix).
The MACS2 software was used for peak identification with data from input as controls and default parameters.
Genome_build: mm9
Supplementary_files_format_and_content: The bedGraph files, which were generated by MACS2 software with default parameters, report the number of ChIP-Seq sequencing reads in a genomic interval (normalized to a read depth of 1 million reads).
|
| |
|
| 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) |
| GSE71472 |
Effect of DNMT3A R882H mutation or WT expression on epigenetic landscapes of hematopoietic stem/progenitor cells with NRAS G12D co-transduction (ChIP-seq) |
| GSE71475 |
Epigenetic perturbations by Arg882-mutated DNMT3A potentiate aberrant stem cell gene expression program and acute leukemia development |
|
| Relations |
| BioSample |
SAMN03941705 |
| SRA |
SRX1122907 |
