|
| Status |
Public on Jul 11, 2016 |
| Title |
Tet2_3aDKO H3K27me3 ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Hematopoietic stem cells (HSC)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: Hematopoietic stem cell
genotype/variation: Tet2 and Dnmt3a double knockout
chip antibody: H3K27me3 (Millipore, catalog# 07-449)
|
| Treatment protocol |
The HSCs used in this study is from genetic engineering mouse model which Tet2 is knocked-out or both Tet2 and Dnmt3a is knocked-out.
|
| Growth protocol |
bone marrow was isolated from Tet2 KO and Dnmt3a-Tet2 DKO mice and transplanted into CD45.1 receipient mice for 2 serial transplantation. Donor derved HSC (CD45.2+ CD150+ CD48- Sca1+ ckit+ Lin-) are isolated by FACS after the sacrifice of mice.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HSCs purified as indicated above were sorted and crosslinked with 1% formaldehyde at room temperature (RT) for 10 min, and the reaction was stopped by 0.125M glycine at RT for 5 min. Cross-linked cells were lysed and sonicated to 200-500 bp fragments (Bioruptor, Diagenode). ChIP-qualified antibodies (H3K27me3 Millipore 07-449) were added to the sonicated chromatin and incubated at 4°C overnight. Following this, 10 μl of protein A magnetic beads (Dynal, Invitrogen) previously washed in RIPA buffer were added and incubated for an additional 2 hours at 4°C. The bead: protein complexes were washed three times with RIPA buffer and twice with TE buffer. Following transfer into new 1.5 ml collection tube, genomic DNA was eluted for 2 hours at 68°C in 100 µl Complete Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 µg/ml proteinase K), and combined with a second elution of 100 µl Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl) for 10 min at 68°C. ChIPed DNA was purified by MinElute Purification Kit (Qiagen) and eluted in 12 µl elution buffer. ChIP-seq library is constructed using Rubicon Genomics ThruPLEX DNA-seq Kit following the manufacture's protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
technical replicate of biological replicate 1
|
| Data processing |
use bowtie2/2.2.7 to align chip sequencing reads to the mouse genome mm9
use MACS2 to identify the peaks of CMS
Genome_build: mm9
Supplementary_files_format_and_content: BED files include chromosome, start, end, peak name, peak enrichment -log(q-value)
|
| |
|
| Submission date |
Jul 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jianzhong Su |
| E-mail(s) |
jianzhongsu82@gmail.com
|
| Phone |
8322902241
|
| Organization name |
Baylor college of Medicine
|
| Street address |
1 Baylor Plaza
|
| City |
HOUSTON |
| State/province |
TEXAS |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE72148 |
DNMT3A and TET2 Compete and Cooperate to Repress Differentiation Lineage-Specific Factors in Hematopoietic Stem Cells |
|
| Relations |
| BioSample |
SAMN05371077 |
| SRA |
SRX1924143 |
