|
| Status |
Public on Apr 30, 2015 |
| Title |
ATAC-Seq from induced pluripotent stem cells |
| Sample type |
SRA |
| |
|
| Source name |
mouse induced pluripotent stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: A12 (Mbd3 flox/-)
transgene: A12 (Mbd3 flox/-)
|
| Treatment protocol |
IES9 sample was generated by addting dox and 4OHT to the medium from day 1- 9
|
| Growth protocol |
Intermediate ES like cells (IES 9 cells): Nanog-CreER Reporter MEFs were plated at day 0 in MEF medium: DMEM supplemented with 1% L-Glutamine, 1% Non-essential amino acids, 1% penicillin/streptomycin and 10% FBS. On day 1 medium was changed to KO-DMEM with supplemented 15% FBS and 5% KSR with added JAK1 inhibitor (Calbiochem 420099, 0.5ϻM) for 6 days, followed by switching to 1% FBS and 14% KSR and JAK1 inhibitor for 3 days. The medium was supplemented with 1% L-Glutamine, 1% Non-essential amino acids, 1% penicillin/streptomycin and 0.1 mM β-mercaptoethanol. Doxycycline hyclate (Dox) and 4-Hydroxytamoxifen (4OHT) were added to the medium at day 1 and continuously kept in the medium until day 9.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nanog-CreER reporter MEFs were reprogrammed using OSKM-TD protocol until the end of OSKM induction step, at which point tdTomato+ intermediate cells were sorted and applied for ATAC sequencing. Briefly, 50,000 sorted cells were centrifuged at 500g for 3 minutes, followed by a wash using 50 μL of cold PBS and centrifugation at 500g for 3 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. Next, the pellet was re-suspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 minutes at 37 °C and immediately put on ice. Directly afterwards, the sample was purified using a Qiagen MinElute kit.
Following purification, the library fragments were amplified using custom Nextera PCR primers 1 and 2 for a total of 12 cycles. Following PCR amplification the libraries were purified using a Qiagen MinElute Kit.
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
We used Illumina CASAVA 1.8.2 software to generate fastq files.
Reads were aligned to mm10 mouse genome using Bowtie2 with the parameter -X2000 (allowing fragments up to 2 kb to align). Duplicated aligned reads were removed using Picard MarkDuplicates tool.
To identify chromatin accessibility signal we considered only short reads (≤ 100bp) that correspond to nucleosome free regions. To detect and separate accessible loci in each sample, we used MACS version 1.4.2-1 with --call-subpeaks flag (PeakSplitter version 1.0).
Genome_build: mm10
Supplementary_files_format_and_content: bed format, significant accessibility signal peaks (MACS output)
|
| |
|
| Submission date |
Mar 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Noa Novershtern |
| E-mail(s) |
noa.novershtern@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics
|
| Street address |
Weizmann Institute
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL18480 |
| Series (2) |
| GSE67298 |
Frequent and Transient Acquisition of Pluripotency During Somatic Cell Trans-Differentiation with iPSCs Reprogramming Factors (ATAC-Seq) |
| GSE67299 |
Frequent and Transient Acquisition of Pluripotency During Somatic Cell Trans-Differentiation with iPSCs Reprogramming Factors |
|
| Relations |
| BioSample |
SAMN03447987 |
| SRA |
SRX969071 |
