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| Status |
Public on Sep 06, 2019 |
| Title |
TKO 24h A: H3K27ac KLF4 KO ChIP-seq |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cells (V 6.5)
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cells (V 6.5)
culture: cultured in 2i condition
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| Treatment protocol |
V6.5 cells were infected using lentiviruses harboring the c3GIC9 vector76 (TRE3G-Cas9-P2A-GFP-PGK-Puro-IRES-rtta) containing gRNA/s targeting either KLF4 only or KLF2, KLF4 and KLF5 in tandem. Following infection cells were selected using Puromycin (LifeTech K210015) and clonal populations were manually picked. Expression of CRISPR-Cas9 from these stable cell lines was induced by addition of Doxycycline for 24hrs (1:1000 dilution of 2mg/ml stock) and KO efficiency in each clonal population was verified by WB: KLF4 (R&D, AF3158) KLF5 (R&D AF3758) KLF2 (Novus biologicals, NBP6181) ESRRB (PPMX, PPH6705) NANOG (Bethyl laboratories, A300-397A) ACTIN (abcam, ab49900). Successful KO clones were then used for subsequent experiments (qPCR, ChIP-seq, RNA-seq, 3C and HiChIP) after induction with doxycycline for the indicated time points (ESC TKO 0 hours and ESC TKO 24 hours).
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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, with or without the presence of 2i (1uM MEKinhibitor (Stemgent 04-0006) and 3uM GSK3 inhibitor (Stemgent 04-0004)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChiP-seq: iPSC and ESC 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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| Description |
V6.5 cells were infected using lentiviruses harboring the c3GIC9 vector76 (TRE3G-Cas9-P2A-GFP-PGK-Puro-IRES-rtta) containing gRNA/s targeting either KLF4 only or KLF2, KLF4 and KLF5 in tandem. Expression of CRISPR-Cas9 from these stable cell lines was induced by addition of Doxycycline for 24 hours.
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| Data processing |
Alignment of the ChIP-seq data was performed with bowtie2 algorithm (--very-sensitive option). Filtering of mitochondial reads, duplicate and low quality mapped reads was performed with the use of samtools and bedtools while peak calling was done with MACS2 (--nomodel –shift 37 –extsize 73 -p 1e-3). For all biological replicates we selected the common peaks
Genome_build: mm10
Supplementary_files_format_and_content: Only peaks common between the replicates were used for downstream analysis.
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| Submission date |
Apr 08, 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 |
GPL17021 |
| Series (2) |
| GSE113431 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency |
| GSE129494 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency [ChIP-seq] |
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| Relations |
| BioSample |
SAMN11360296 |
| SRA |
SRX5654587 |
| 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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