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Sample GSM842252 Query DataSets for GSM842252
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

Supplementary file Size Download File type/resource
GSM842252_HOXA9_MM0338_3l_FP2_mm8_xset150_dupsN_ht12.sub.wig.gz 305.4 Kb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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