|
| Status |
Public on Dec 04, 2011 |
| Title |
Hoxa replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
myeloblastic cell lines transformed with an MSCV-based retrovirus expressing HA-Hoxa9 and Flag-Meis1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: myeloblastic cell lines transformed with an MSCV-based retrovirus expressing HA-Hoxa9 and Flag-Meis1
chip antibody: anti-HA
chip antibody vendor: Abcam
chip antibody catalog#: ab9110
|
| Growth protocol |
Bone marrow cells were harvested from 5-Fluorouracil treated female 6-8 week old C57BL/6 mice and transformed into myeloblastic cell lines with an MSCV-based retrovirus expressing HA-tagged or untagged Hoxa9 or Meis1 (HM2 cells). For conditional activation of Hoxa9, cells were transformed with Hoxa9 fused to a modified estrogen receptor ligand-binding domain (Hoxa9-ER). Cells were cultured in Iscove’s Modified Dulbecco’s Medium with 15% fetal bovine serum (Stem Cell Technologies) and penicillin/streptomycin.IL-3 (R&D) was added to media; alternatively cells were transduced with an IL-3-expressing retroviral vector (pMFGmIL3, obtained from RIKEN DNA Bank with consent of Dr. Hirofumi Hamada); Hoxa9-ER cells were also supplemented with 4-OHT (Sigma). Hoxa9 is required for continued mouse hematopoietic progenitor (MHP) survival, so positive selection of transduced clones was not necessary; double Hoxa9/Meis1 transductants were selected by fluorescence-activated cell sorting (bicistronic Meis1+GFP expression using MigR1 vector; a gift from Dr. Warren Pear).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A total of 150 million cells were cross-linked sequentially with disuccinimidylglutarate (45 min RT) and 1% formaldehyde (15 min RT). Hoxa9 and Meis1 immunoprecipitation was performed with anti-HA antibody (Abcam) pre-conjugated to Protein G magnetic beads (Dynal/Invitrogen). 4-hour incubation (4°C with gentle rotation) was followed by washes using Low Salt, High Salt, LiCl, and Tris-EDTA buffers (Upstate/Millipore). Immunoprecipitateswere eluted with 0.1% SDS/0.1M NaHCO3 and cross-links were reversed overnight at 65° in 0.2M NaCl. DNA was RNAse-treated and column purified (Qiaquick, Qiagen). For ChIP-Seq, size selection and sequencing were performed as described previously (Robertson et. al 2007)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against Hoxa9
MM0338
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse (mm8; Feb 2006) genomes using the Illumina Genome Analyzer Pipeline. We determine for each profile a threshold peak height as the smallest height that corresponds to FDR < 0.001 for peaks of that height.
|
| |
|
| Submission date |
Dec 04, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jay Hess |
| E-mail(s) |
jayhess@med.umich.edu
|
| Phone |
734-763-6384
|
| Fax |
734-763-4782
|
| URL |
http://www.pathology.med.umich.edu/faculty/Hess/index.html
|
| Organization name |
University of Michigan
|
| Department |
Pathology
|
| Street address |
1301 Catherine
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE33509 |
Identification and Characterization of Hoxa9 Binding Sites in Hematopoietic Cells |
| GSE33518 |
Identification and characterization of Hoxa9 binding sites in hematopoietic cells |
|
| Relations |
| SRA |
SRX110442 |
| BioSample |
SAMN00761831 |
