|
| Status |
Public on Jun 18, 2019 |
| Title |
O4SK_d1_rep2ATAC |
| Sample type |
SRA |
| |
|
| Source name |
OG2MEF
|
| Organism |
Mus musculus |
| Characteristics |
cell type: reprogramming cells
day of reprogramming: Day1
|
| Treatment protocol |
NA
|
| Growth protocol |
OG2-MEF cells were transduced twice by pMX-Sox2, Klf4 along with Oct4 or Oc6 or engineered Oct4 (YR or Oct4defSox2) retroviruses produced using PlatE cells in DMEM + 10% FBS media supplemented with 1x NEAA and Glutamax. After second viral transduction, the medium was switched to highly efficient chemically defined (iCD1) reprogramming medium. The iCD media switch day was defined as day 0 and cells were further cultured with daily media change.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A total of 50,000 cells (reprogrammed with O4SK, O6SK and O4defS2SK retroviruses) were collected at days 1 and 5 of reprogramming in replicates. Cells were washed once with 50 μL of cold PBS; centrifuged at 500 x g for 5 min at 4°C and cell pellets were resuspended in 50 μL cold ATAC lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) by slowly pipetting up and down and immediately spun down at 500 x g for 10 min at 4°C to collect nuclei. Nuclei were washed in 1x PBS and subsequently re-suspended in 50 μL transposition reaction mix (25 μL 2 x TD reaction buffer, 22.5 μL nuclease-free water, 2.5 μL Tn5 transposase) of Nextera DNA library preparation kit (FC-121-1030, Illumina). Samples were incubated at 37°C for 30 min and DNA was isolated using minElute Kit (Qiagen). The transposed DNA was then amplified with custom primers as described for 1 cycle of 72°C for 5 min, 98°C for 30 sec followed by 5 cycles of 98°C for 10 sec, 63°C for 30sec, 72°C for 1 min. To determine the suitable number of cycles required for next round of PCR the library was assessed by quantitative PCR. Libraries qualities were assessed using Bioanalyzer high sensivity DNA analysis kit (Agilent) followed by paired-end sequencing with the length of 150 nucleotides using Hiseq X10 (illumina) at Annoroad Gene Technology (http://en.annoroad.com/).
The libraries were generated using the Nextera DNA Library Prep kit (Illumina)
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
fastqc
Read were aligned to mouse genome assembly (mm10) using bowtie2 (--very-sensitive --end-to-end --no-unal --no-mixed -X 2000).
Low quality mapped reads were removed using samtools (view –q 30).
PCR duplicates were removed using samtools (rmdup) to keep uniquely mapped reads.
The BAM files of time point replicates of each sample were merged with samtools (merge) prior to peak calling with MACS2 (-g mm -f BAMPE).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig file were generated with genomeCoverageBed followed by bedGraphToBigWig.
|
| |
|
| Submission date |
Sep 04, 2018 |
| Last update date |
Jun 19, 2019 |
| Contact name |
Veeramohan Veerapandian |
| E-mail(s) |
veeramohan.v@mpi-muenster.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Biomedicine
|
| Department |
Department Tissue Morphogenesis
|
| Street address |
Röntgenstr. 20
|
| City |
Munster |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
48149 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21273 |
| Series (2) |
| GSE103980 |
Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2 |
| GSE119396 |
Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2 [ATAC-Seq] |
|
| Relations |
| BioSample |
SAMN09949873 |
| SRA |
SRX4636336 |
