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| Status |
Public on Sep 06, 2019 |
| Title |
ES-Tbx3_enhancer_rep2 |
| Sample type |
SRA |
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| Source name |
ES-Tbx3_enhancer
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| Organism |
Mus musculus |
| Characteristics |
cell tye: Embryonic stem cells
culture condition: cultured in 2i condition
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| Treatment protocol |
N/A
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| Growth protocol |
MEF were grown in low oxygen incubator with DMEM media, V6.5 were grown in KO-DMEM media in 2i conditions (TKO samples-with or without dox for 24 hrs) , MEF at different times of reprogramming were grown in the presence of dox and ascorbic acid
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were collected with trypsin and fixed with 1% formaldehyde
Cell pellets were lysed in 1ml Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA; 1x complete protease inhibitor, 0.5% NP-40, 1% triton) and incubated on ice for 15 min. The samples were centrifuged at 2500xG for 5min at 4°C and the pellet was then resuspended in 360µl milli-Q, 60µl 10X DpnII restriction buffer and 15ul 10%SDS. After 1 hour incubation at 37ºC, 150ul of 10% Triton was added and samples were incubated again at 37ºC for 1 hour. 4ul DpnII enzyme (#R0543M, NEB) were added and samples were incubated at 37ºC over night while shaking in a thermomixer (9000rpm). After confirming the digestion efficiency, the enzyme was inactivated by adding 80ul 10% SDS and incubating at 65 ºC for 30 mins. The digested samples were then diluted with 4860ul Milli-Q, 700ul ligation buffer (500mM Tris pH 7.5, 100mM DTT, 100mM MgCl2,10mM ATP), and 750ul of Triton and incubated at 37ºC for 1 hour. Then 2ul Ligase (NEB M0202M) were added and samples were incubated over night at 16 ºC. Next morning, after testing the ligation efficiency, we reversed the crosslinks by adding 30ul of proteinase K (10mg/ml) and incubating over night at 65ºC. Subsequently the RNA was removed using 30ul of RNase A (10mg/ml) for 45mins at 37ºC. Extensive phenol/chloroform extraction was followed by EtOH precipitation and two washes with 70% EtOH. The pellets were dissolved in 150ul 10mM Tris pH 7.5 by incubating at 37 ºC. We then added 50ul 10x buffer B (Fermentas), 5ul Csp6I (Fermentas, ER0211) and 299ul milli-Q water and samples were digested at 37ºC over night. After determining digestion efficiency, the restriction enzyme was inactivated by incubating the tubes at 65ºC for 25 mins. Samples were diluted in 12ml milli-Q, 3ul ligase (NEB, M0202M) and 1.4ml 10X ligation buffer (500mM Tris pH7.5, 100mM DTT, 100mM MgCl2, 10mMATP) and incubated over night at 65ºC. Following phenol/chloroform and EtOH precipitation the pellets were dissolved in 300ul 10mM Tris pH7.5 and DNA was further purified using 4 Zymo columns per sample (Zymo, D4014). Each sample was eluted in 200ul total of 10mM Tris pH7.5
PCR-amplification using the KAPA HiFi enzyme (KAPA biosystem, 07958927001). Four PCR reactions were combined per sample, following column purification using the ZYMO kit (Zymo, D4014).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
processed data file: Tbx3_enhancer_cpm.tsv
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| Data processing |
library strategy: 4C-seq
Read trimming: seqtk. Known adapter sequences were trimmed off the reads.
Read alignment: bowtie 1.0.0. Reads were mapped against redcued mm10 reference sequence using bowtie. Reduced reference genome only consists of sequences adjacent to DpnII cut-sites. Multi-mapped reads were discarded.
Read counts: custom script. The genome was binned in successively overlapping 10kb bins, with each bin overlapping by 9kb with the next bin. Read-mates were counted per bin if second mate mapped within +/-5kb around virtual viewpoint.
Normalization: edgeR. Raw read counts were normalized with R library edgeR function cpm.
Genome_build: mm10
Supplementary_files_format_and_content: custom tsv: counts-per-million (CPM) per 10kb bin
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| Submission date |
Jul 30, 2019 |
| Last update date |
Oct 10, 2023 |
| Contact name |
Effie Apostolou |
| E-mail(s) |
efa2001@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Street address |
1161 York Avenue, Apt 8A
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE113431 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency |
| GSE135124 |
KLF4 binding during reprogramming is linked to enhancer rewiring and is critical for the architecture and regulation of enhancer hubs [4C-Seq] |
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| Relations |
| BioSample |
SAMN12412443 |
| SRA |
SRX6616790 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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