|
| Status |
Public on Dec 21, 2012 |
| Title |
empty-vector_flagChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow cells_empty vector_flagChIP
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: hematopoietic stem/progenitor cells
transduced with: empty vector
culture age: 7 days
chip antibody: M2 Flag-agarose beads
chip antibody vendor: Sigma
chip antibody cat. #: A2220
chip antibody lot #: 069K6094
|
| Growth protocol |
Bone marrow cells isolated from C57BL/6 mice, injected with 5FU five days prior to sacrifice, were retrovially transduced with empty vector, flag-Cbx7 and flag-Cbx8. Transduced GFP+ cells were sorted by FACS and cultured in StemSpan (StemCell Technologies) supplemented with 10% fetal bovine serum, 300 ng/mL polyethylene glycol–complexed recombinant rat stem cell factor (Amgen), 20 ng/mL recombinant murine interleukin-11 (R&D Systems), 1 ng/mL Flt3 ligand (Amgen), and penicillin and streptomycin for 7 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation assays (ChIP) of transduced bone marrow cells were in essence performed and analyzed as described previously (Frank S.R. etal., Genes Dev. 15, 2069-82 , 2001) with some minor adjustments. Flag-ChiP using M2 Flag-agarose beads was performed in IP150 buffer and complexes were washed 4x in 500mM wash buffer (1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA pH 8.0 ,20mM Tris-HCl pH 8.0) and the final wash step was performed in TE buffer. DNA samples obtained from the ChIP assays were adaptor-ligated and amplified (New England Biolabs kit E6040).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Sample 1
|
| Data processing |
Base calling were done using Illumina’s analysis Pipeline in two stages with: RTA Version: 1.09 Illumina machine, and configureBclToQseq.py version 1.9.0, on the unix server.
The default Illumina pipeline quality filter is used, which uses a threshold of CHASTITY >= 0.6. Chastity for a given base call is defined as "the ratio of the highest of the four (base type) intensities to the sum of highest two."
Duplicate sequence reads were removed to avoid PCR bias
Base calling: Bowtie alignment algorithm for Illumina sequence format (version 1.1.2)
Reads were mapped to the mouse genome (NCBI37/mm9)
Peak detection was performed using MACS1.0.1 (Genome size 1.87e+9, tag size 36bp, cutoff peak detection 1e-5).
Empty vector was used for negative control ChIP-signal, in addition a cross background analysis was performed in which Cbx7 ChiP signal served as control for Cbx8 peakcalling and the other way around.
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using Bowtie
|
| |
|
| Submission date |
Mar 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Leonid Bystrykh |
| E-mail(s) |
l.bystrykh@umcg.nl
|
| Organization name |
University Medical Center Groningen
|
| Department |
European Research Institute for the Biology of Ageing - ERIBA
|
| Lab |
Ageing Biology and Stem Cells
|
| Street address |
A. Deusinglaan 1
|
| City |
Groningen |
| State/province |
Groningen |
| ZIP/Postal code |
9713 AV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE36658 |
Polycomb Cbx orthologs mediate the balance between Hematopoietic Stem Cell self-renewal and differentiation |
|
| Relations |
| SRA |
SRX130469 |
| BioSample |
SAMN00829215 |
