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Sample GSM1543574 Query DataSets for GSM1543574
Status Public on Jan 01, 2015
Title SclKO-iScl_ES_Scl_INP
Sample type SRA
 
Source name ES cells
Organism Mus musculus
Characteristics cell type: ES cells
cell-line: SclKO-iScl mESC
treatment: 48h 1ug/ml doxycycline
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.
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.
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Nov 12, 2014
Last update date May 15, 2019
Contact name Tõnis Org
E-mail(s) toniso@ut.ee
Phone 372-52-11484
Organization name University of Tartu
Department Institute of Molecular and Cell Biology
Lab Biotechnology
Street address Riia 23
City Tartu
ZIP/Postal code 51010
Country Estonia
 
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
Relations
BioSample SAMN03175437
SRA SRX757573
Named Annotation GSM1543574_SclKO-iScl_ES_Scl_INP.bw

Supplementary file Size Download File type/resource
GSM1543574_SclKO-iScl_ES_Scl_INP.bw 59.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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