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| Status |
Public on Sep 06, 2019 |
| Title |
EC-EA-3702-KLF4_DAY9_1 |
| Sample type |
SRA |
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| Source name |
DAY9
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 x 129
genotype: OKSM Col1a1/M2rtTA ROSA26
Stage: DAY9
medium: ESC medium with 1ug/ml doxycycline and 50ug/ml ascorbic acid
marker: sorted for SSEA+
antibody: KLF4 R&D #3162
batch: EC-EA-3702
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| Growth protocol |
Mouse ES V6.5 were cultured on irradiated feeder cells in KO-DMEM media (Invitrogen) supplemented with 15% heat-inactivated fetal bovine serum, GlutaMAX, penicillin-streptomycin, non-essential amino acids, β-mercaptoethanol and 1000 U/ml LIF (ESC medium). All V6.5 samples for HiCHIP and two replicates of ChIPseq were cultured in the presence of 2i (1uM MEKinhibitor (Stemgent 04-0006) and 3uM GSK3 inhibitor (Stemgent 04-0004)). Mouse embryonic fibroblasts (MEFs) were isolated from a "reprogrammable" mouse harboring a polycystronic OKSM cassette in the Col1a1 locus and M2rtTA in the Rosa26 locus. Cells were reprogrammed in the presence of 1ug/ml doxycycline and 50ug/ml ascorbic acid and cultured in ES medium as described above.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked in 1% formaldehyde at RT for 10 minutes and quenched with 125mM glycine for 5 mins at RT. 50 million cells were used for KLF4 ChIPs and 10 million for H3K27acetylation ChIP. Cell pellets were washed twice in PBS and resuspended in 400ul lysis buffer (10mM Tris pH8, 1mM EDTA, 0.5% SDS) per 20 million cells. Cells were sonicated in a bioruptor device (30 cycles 30sec on/off, high setting) and spun down 10 minutes at 4°C at maximum speed. Supernatants were diluted 5 times with dilution buffer (0.01%SDS, 1.1% triton,1.2mM EDTA,16.7mM Tris pH8, 167mM NaCl) and incubated with the respective antibody (2-3ug/10M cells) (KLF4 R&D #3158, H3K27ac ab4729) O/N with rotation at 4°C. Next day, protein G Dynabeads (ThermoScientific) preblocked with BSA protein (100ng per 10ul Dynabeads) were added (10ul blocked Dynabeads per 10 million cells) and incubated for 2-3 hours at 4°C. Beads were immobilized on a magnet and washed twice in low salt buffer (0.1% SDS,1% triton, 2mM EDTA, 150mM NaCl, 20mM Tris pH8), twice in high salt buffer (0.1% SDS,1% triton, 2mM EDTA, 500mM NaCl, 20mM Tris pH8), twice in LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris pH8) and once in TE. DNA was then eluted from the beads by incubating with 150ul elution buffer (1% SDS, 100mM NaHCO3) for 20 minutes at 65°C (vortexing every 10min). Supernatants were collected and reverse-crosslinked by incubation at 65°C O/N in presence of proteinase K. After RNase A treatment for 1hr at 37°C, DNA was purified using the minElute kit (Qiagen).
6-10ng of immunoprecipitated material was used for ChIP-seq library preparation using the KAPA Hyper prep kit (KAPA Biosystems). Libraries were sequenced on an Illumina HiSeq 2500 platform on SE50 mode.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads were trimmed for residual adapters (cutadapt version 1.8.1).
Alignment to the mouse reference genome version mm10 was performed using standard parameters, permitting maximum one mismatch in seed alignment (Bowtie version 2.2.5).
Reads marked as positional duplicates or falling into mouseENCODE blacklisted genomic regions (liftOver to mm10; Consortium, 2012) were filtered out.
Coverage tracks in RPKM were generated from alignments using bamCoverage (V2.3.6).
ChIP-seq peaks (enrichment of signals over background) were called at 10^-2 (MACS version 2.1.1), and peaks detected in more than half of biological replicates were retained for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files for coverage tracks; BED files for peak coordinates.
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| Submission date |
Apr 20, 2018 |
| 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 |
GPL17021 |
| Series (2) |
| GSE113429 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency [ChIP-seq] |
| GSE113431 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency |
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| Relations |
| BioSample |
SAMN08963153 |
| SRA |
SRX3979858 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3106275_EC-EA-3702-KLF4_DAY9_1.bed.gz |
467.0 Kb |
(ftp)(http) |
BED |
| GSM3106275_EC-EA-3702-KLF4_DAY9_1.bw |
159.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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