|
| Status |
Public on Jan 25, 2019 |
| Title |
ATAC BRACHYURY rep1 |
| 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 BRACHYURY-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 |
SAMN10032310 |
| SRA |
SRX4669278 |
