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| Status |
Public on Jan 01, 2015 |
| Title |
SclKO-iScl_ES_Scl_rep1 |
| Sample type |
SRA |
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| Source name |
ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES cells
cell-line: SclKO-iScl mESC
treatment: 48h 1ug/ml doxycycline
chip antibody: Scl/Tal1
chip antibody vendor: Santa Cruz
chip antibody cat. #: c-12984X
chip antibody lot #: A1613
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| Treatment protocol |
ES cells were treated 48h with 1ug/ml doxycycline. ES cells were differentiated into Flk1+ cells by 2 days of culture in pre-EB media (IMDM with the addition of L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M monothiolglycerol (MTG), 15% of FBS and 10ng/ml LIF; followed by plateing 100 000 cells per well to low attachment 6-well plates (Costar) in EB media ((IMDM with the addition of 15% of FBS, L-glutamine, 1% of penicillin and streptomycine, 1.5 x 10-4 M MTG, 3mg/ml human transferrin (Roche) and 2.5mg/ml ascorbic acid (Sigma) and clulturing the cells for 4 days. At day 2 of EB culture the cells were treated for 48h with 1ug/ml doxycycline.
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| Growth protocol |
Standard ES cell culture media with DMEM (Cellgro), 15% serum (Hyclone or Omega) and 10ng/ml LIF (Millipore) and gelatin coated dishes were used to maintain SclKO-iScl ES cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized, washed 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 |
Nov 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tõnis Org |
| E-mail(s) |
[email protected]
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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 |
SAMN03175438 |
| SRA |
SRX757571 |
| Named Annotation |
GSM1543572_SclKO-iScl_ES_Scl_rep1.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1543572_SclKO-iScl_ES_Scl_rep1.bw |
58.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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