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Sample GSM2176273 Query DataSets for GSM2176273
Status Public on Apr 30, 2019
Title Day5_CPC_ATAC-seq-Rep1
Sample type SRA
 
Source name Cardiac progenitors
Organism Mus musculus
Characteristics cell type: ES cell derived cardiac progenitor cells
passages: 16-20
strain: C57BL/6
Treatment protocol N/A
Growth protocol E14 Tg(Nkx2–5-EmGFP) ES cells were maintained on mitomycin treated MEF in Knockout DMEM medium containing 4.5 mg/ml D-glucose, supplemented with 10% serum replacement, 2 mM L-Glutamine, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1,000 U/ml of leukemia inhibitory factor. For directed cardiomyocyte differentiation, ES cells were grown on gelatin in Neurobasal medium: DMEM/F12 (1:1) medium supplemented with 2,000 U/ml LIF and 10 ng/ml BMP4 for 2 days and differentiated by aggregation in low attachment bacterial dishes at a cell density of 75000 cells/ml in IMDM: F12 medium (3:1). After 48h aggregates were dissociated and re-aggregated in the presence of 5 ng/ml Activin A, 5ng/ml VEGF and 0.2ng/ml BMP4. 40h following the second aggregation, aggregates were dissociated and plated as a monolayer in Stempro-34 medium supplemented with Stempro34 nutrient supplement, L-Ascorbic Acid and 5ng/ml VEGF, 10ng/ml bFGF, and 25ng/ml FGF10 growth factors.
Extracted molecule genomic DNA
Extraction protocol ES cell derived cardiac progenitors were trypsinized and washed in PBS. 100,000 cells were resuspended in 50 uL cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630) and used for ATAC Library preparation using Tn5 Transposase from Nextera DNA Library Prep Kit (Illumina #15028211). After centrifugation (10 min, 500 g at 4 °C), cell pellet was resuspended in transposition reaction mix (25 µl TD-Buffer, 2.5 µl Tn5, 22.5 µl water) and incubated for 30 min at 37 °C with gentle mixing. Immediately following the transposition reaction, purification was carried out using MinElute PCR Purification Kit (Qiagen #28004).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Ion Torrent Proton
 
Data processing Library strategy: ATAC-Seq
Visualization of raw reads was done using FastQC
ATAC-Seq reads were mapped with bowtie2 to mm9 mouse genome. Samtools view (-F 1804 -f2) was used to remove unmapped, mate unmapped, not primary aligned reads. The MarkDuplicates.jar from Picard 1.136 was use to remove PCR artefacts. Peaks calling was performed with MACS14 60 (p1e5 --nomodel and --shiftsize 100), and peaks overlapping blacklist defined by ENCODE with the help of Bedtools v2.15 (intersect -wa)
BamCoverage function of deepTools2 62 (-b 20 -smooth 40 -e 150) was used to create normalized bigwig files (reads per genome coverage, RPGC)
Genome_build: mm9
Supplementary_files_format_and_content: Day5_CPC_Isl1_control_ATAC_Seq_merge.bw
Supplementary_files_format_and_content: Day5_CPC_control_ATAC_seq_peaks_annot.bed: Peaks file.
 
Submission date May 24, 2016
Last update date May 15, 2019
Contact name Gergana Dobreva
E-mail(s) Gergana.Dobreva@medma.uni-heidelberg.de
Organization name Medical Faculty Mannheim/University of Heidelberg
Department Cardiovascular Genomics and Epigenomics
Lab AG Dobreva
Street address Ludolf-Krehl-Str. 7-11
City Mannheim
State/province Baden Württemberg
ZIP/Postal code 68167
Country Germany
 
Platform ID GPL18635
Series (2)
GSE80383 Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate
GSE81815 Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate [CPC_ATAC-seq]
Relations
BioSample SAMN05171463
SRA SRX1797665

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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