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Sample GSM3383693 Query DataSets for GSM3383693
Status Public on Jan 25, 2019
Title ATAC OCT4 rep2
Sample type SRA
 
Source name NIH-3T3
Organism Mus musculus
Characteristics cell line: NIH-3T3
cell type: mouse fibroblasts
Treatment protocol NIH-3T3 cells were treated with 500 ng/ml of Doxycycline 48 hours before the experiment. 50'000 cells were collected.
Growth protocol NIH-3T3 cells were routinely cultured at 37°C and 5% CO2 in DMEM, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown in 100 mm cell culture dishes up to a confluence of 90% and split 1:6 every 3-4 days.
Extracted molecule genomic DNA
Extraction protocol Cells were pelleted and washed with 1X ice cold PBS at 800g for 5 min. Cells were gently resuspended in 50 μl of ice-cold ATAC lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40), and immediately pelleted at 800g for 10 min at 4°C. To transpose open chromatin regions, cells were resuspended in 50 μl of transposition reaction mix containing 0.5 μM of Tn5 transposase (in-house prepared) in TAPS-DMF buffer (10 mM TAPS-NaOH, 5 mM Mgcl2, 10% DMF) and incubated at 37°C for 30 min. The transposed DNA was purified using a DNA purification kit and eluted in 12 μl of water.
A 65 μl PCR reaction was setup with 10 μl of transposed DNA, 0.5 μM of forward primer Ad1_noMX, 0.5 μM of multiplexing reverse primer Ad2.x (Buenrostro et al. 2013), 0.6x SYBR Green I, and 1x PCR Master Mix. The samples were thermocycled at 72°C for 5 minutes, 98°C for 30 s, followed by 12 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. The amplified ATAC libraries were purified using a DNA purification kit and size selected using Agencourt AMPure beads (0.55X unbound fraction followed by 1.2X bound fraction).
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description Overexpression of OCT4-HA
Data processing Reads were aligned to the mouse reference genome mm10 using STAR 2.5.3a with settings ‘-- alignMatesGapMax 2000 --alignIntronMax 1 --alignEndsType EndtoEnd -- outFilterMultimapNmax 1’
Duplicate reads were removed with Picard and reads not mapping to chromosomes 1-19, X, or Y were removed

Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated by merging replicate bam files with SAMtools followed by the deepTools functions bamCoverage (with setting --normalizeUsingRPKM). log2-ratio bigWig files were generated using the deepTools function bamCompare based on merged replicates for each TF overexpression sample over merged replicates for control (rtTA3G). on merged duplicates
 
Submission date Sep 11, 2018
Last update date Jan 25, 2019
Contact name David Michael Suter
Organization name École polytechnique fédérale de Lausanne
Department Institute of Bioengineering
Lab UPSUTER
Street address EPFL SV-IN Station 19
City Lausanne
ZIP/Postal code 1015
Country Switzerland
 
Platform ID GPL19057
Series (2)
GSE119781 Mitotic chromosome binding predicts transcription factor properties in interphase [ATAC-Seq]
GSE119784 Mitotic chromosome binding predicts transcription factor properties in interphase
Relations
BioSample SAMN10032353
SRA SRX4669293

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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