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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 07, 2024 |
| Title |
JM8.N4 mESC, clone A ∆ZFP143/∆CTCF 3h, CTCF ChIP-seq, rep. 2 |
| Sample type |
SRA |
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| Source name |
JM8.N4
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| Organism |
Mus musculus |
| Characteristics |
cell line: JM8.N4
cell type: Mouse embryonic stem cell
spike-in reference_organism: Homo sapiens
spike-in cell_line: HEK293T
chip antibody: CTCF
antibody info: Millipore-Sigma (#07-725)
genotype: Clone A, Homozygous Rosa26-OsTIR1(F74G), Homozygous ZFP143-mAID2-HaloTag-V5, Homozygous FLAG-SNAPftag-FKBP12(F36V)-CTCF
treatment: 100uM 5-Ph-IAA/300nM HaloPROTAC3/100nM dTAG-13
time: 3 hours
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| Treatment protocol |
For depletion of ZFP143 in the clone A and clone B cell lines, the cells were incubated in media containing 100 μM 5-Ph-IAA (MedChemExpress #HY-134653) and 300 nM HaloPROTAC3 (Promega # GA3110), and for depletion of CTCF in the same lines the, the cells were incubated in media containing 100 nM dTAG-13 (Tocris # 6605) for 3 hours. For the depletion of ZFP143 in the clone D cell line, the cells were incubated in media containing 100 nM dTAG-13, and for the depletion of ZNF143 in the clone 30 HEK293T cell line, the cells were incubated in media containing 500 μM IAA for 3 hours.
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| Growth protocol |
mESCs were cultured under 5% CO2 at 37°C under feeder-free conditions. The mESCs were grown in medium consisting of KnockOut DMEM (ThermoFisher #10829-018) supplemented with 15% FBS, 1000 U/mL LIF (homemade), 1 mM MEM Non-Essential Amino Acid Solution (ThermoFisher #11140050), 2 mM GlutaMAX supplement (ThermoFisher #35050061), 100 μg/mL Penicillin-Streptomycin (ThermoFisher #15140122), 0.1 mM 2-mercaptoethanol (ThermoFisher #31350010), 10 μM MEK inhibitor (Tocris #PD0325901), and 3 μM GSK inhibitor (Sigma-Aldrich #SML1046) on plates pre-coated with sterile 0.1% gelatin (Sigma-Aldrich, G1890). The mESCs were passaged every 2 days by dissociation with TrypLE Express Enzyme (ThermoFisher #12605028), and half the medium was replaced daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 mins. Following quenching, chromatin was sonicated and then reverse crosslinked. Naked DNA was further sonicated to the 400-800bp range.
DNA from ZNF143 ChIP, CTCF ChIP, or input chromatin was used to prepare libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), using NEBNext Multiplex Oligos for Illumina for indexing samples. Libraries were amplified with a number of PCR cycles according to the manufacturer's protocol on the thermocycler using KAPA HiFi HotStart ReadyMix (Roche) and purified twice using AMPure XP beads (Beckman Coulter). Purified libraries were quantified using qPCR and pooled in a 1:1 ratio. Following qPCR quantification, libraries were submitted for sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
chip_ctcf_a_dual_r2
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| Data processing |
Basecalls were performed using bcl2fastq v2.20.0.422 for NovaSeq output.
Paired end reads were aligned to a concatenation of the UCSC mm39 and hg38 genomes using bowtie2 v2.2.5 with --no-mixed --no-discordant.
Uniquely aligned reads were filtered for PCR duplicates using sambamba markdup. Reads for each condition were randomly subsambled using normalization factors based on hg38 spike-in. Normalization factors were corrected using the ratio of human to mouse read counts in corresponding input samples.
Assembly: mm39;hg38
Supplementary files format and content: bigwig file has data based on all uniquely aligning non-duplicate reads
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| Submission date |
Feb 21, 2024 |
| Last update date |
Mar 07, 2024 |
| Contact name |
Domenic Narducci |
| E-mail(s) |
domenicn@mit.edu
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biological Engineering
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| Lab |
Anders Hansen
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| Street address |
Building 56 Room 787B, 32 Vassar Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE256226 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function [ChIP-seq] |
| GSE256246 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function. |
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| Relations |
| BioSample |
SAMN40017333 |
| SRA |
SRX23679391 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8091498_A9_Dual_R2_CTCF_mm39_MERGED_rmdup_downsampled.bw |
169.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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