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| Status |
Public on Jan 01, 2015 |
| Title |
Hpc7_H3K27ac |
| Sample type |
SRA |
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| Source name |
Hematopoietic progenitors
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| Organism |
Mus musculus |
| Characteristics |
cell line: HPC7
cell type: Hematopoietic progenitors
treatment: no treatment
chip antibody: H3K27ac
vendor: abcam
chip antibody cat. #: ab4729
chip antibody lot #: GR117011-1
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| Treatment protocol |
non-treated
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| Growth protocol |
HPC7 cells were grown in Iscove´s modified Dulbecco´s media (IMDM) (Gibco), supplemented with NaHCO3, 1.5 x 10-4 M MTG (Sigma),1% of penicillin and streptomycine, 10% of FBS and mouse Stem cell factor (SCF) at 100ng/mL (R&D systems)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells werewashed with PBS and crosslinked with 1% formaldehyde in PBS for 10 min at RT. After PBS washing, cells were resuspended in 400 μl of lysis buffer (1% SDS, 20 mM EDTA and 50 mM Tris-HCl (pH 8.0)) containing protease inhibitors (Roche, Indianapolis, IN), incubated for 10 min on ice and sonicated using Misonix cup-horn sonicator to achieve, on average, 200bp fragments. The lysate was diluted 10 times with ChIP dilution buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA and 16.7 mM Tris-HCl (pH 8.1) and immunoprecipitatied with 4 ug of corresponding antibody overnight at 4 degrees. 20 μl of the lysates were used as input. The complexes were captured using protein A Dynabeads (Invitrogen, Grand Island, NY) and washed twice with the following buffers: low-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1); high-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl); LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and TE (10 mM Tris-HCl and 1 mM EDTA (pH 8.0)). After elution with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% SDS, crosslinks were reversed by overnight incubation at 65°C. Samples were then treated with RNase A for 30 min at 37°C and proteinase K for 2 h at 56°C. DNA was subsequently purified using Qiagen MinElute Columns according to manufacturers instructions. DNA concentration was measured using a Qubit (Invitrogen, Grand Island, NY).
The library for sequencing was constructed using Ovation Ultralow DR Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Debarcoding of the multiplex runs was performed using Unix shell scripts.
Tags were mapped to the mouse genome (mm9) using bowtie v0.12.7 with parameters -v 2 -m 1 -S --best --strata
Peak identification was performed with MACS v1.3.7.1 using default parameters
bedgraph files were created using Homer (Heinz et al., 2010) using -auto parameter converted into bigwig (bedGraphToBigWig) and visualized on UCSC (Kent et al., 2002) genome browser as custom tracks
Scl peaks were mapped to nearby genes within 200kb range from TSS using Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean et al., 2010). Peak intersections and overlaps with differentially expressed genes were performed using Galaxy (Blankenberg et al., 2010) and in house Ubix shell scripts.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph files were created using Homer (Heinz et al., 2010) using -auto parameter converted into bigwig (bedGraphToBigWig) and visualized on UCSC (Kent et al., 2002) genome browser as custom tracks
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| Submission date |
Apr 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tõnis Org |
| E-mail(s) |
toniso@ut.ee
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| Phone |
372-52-11484
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| Organization name |
University of Tartu
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| Department |
Institute of Molecular and Cell Biology
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| Lab |
Biotechnology
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| Street address |
Riia 23
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| City |
Tartu |
| ZIP/Postal code |
51010 |
| Country |
Estonia |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE47082 |
Scl specifies hemogenic endothelium and inhibits cardiogenesis via primed enhancers [ChIP-seq] |
| GSE47085 |
Scl specifies hemogenic endothelium and inhibits cardiogenesis via primed enhancers |
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| Relations |
| BioSample |
SAMN02738938 |
| SRA |
SRX528335 |
| Named Annotation |
GSM1375624_Hpc7_H3K27ac.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1375624_Hpc7_H3K27ac.bw |
58.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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