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| Status |
Public on Mar 07, 2024 |
| Title |
JM8.N4 mESC, clone B ∆ZFP143/∆CTCF 3h, Micro-C, rep. 1 |
| 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
genotype: Clone B, 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 3 mM DSG for 35 minutes, followed by an additional 10 minutes with 1% formaldehyde. Following quenching, nuclei were prepared from fixed cells. Chromatin was digested in situ using MNase, DNA 3' ends were chewed back and biotinylated, and then free DNA ends were proximity ligated. Unligated ends were digested, and chromatin crosslinks were reversed. DNA was purified by the DNA Clean and Concentrator kit (Zymo), and size selected for dinucleosomal fragments on an agarose gel. Following end polishing of DNA fragments, a streptavidin pulldown was used to enrich for ligated dinucleosomal fragments.
Size selected DNA fragments bound to streptavidin coated beads were used to prepare Micro-C 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 7-10 PCR cycles on the thermocycler using KAPA HiFi HotStart ReadyMix (Roche) and purified using AMPure XP beads (Beckman Coulter). Purified libraries were quantified using qPCR and pooled in a 1:1 ratio. Following qPCR quantification, the library pool was submitted for sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
microc_b_dual_r1
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| Data processing |
Assembly: mm39
*library strategy: Micro-C
Basecalls were performed using bcl2fastq v2.20.0.422 for NovaSeq output.
Paired end reads were aligned to the UCSC mm39 genome using bowtie2 v2.2.5 with --local --reorder --very-sensitive-local. Aligned paired end reads were then parsed with pairtools (v0.3.0) parse with --add-columns mapq --walks-policy mask --min-mapq 2 --drop-sam --drop-readid.
Parsed reads were filtered for PCR duplicates and unmapped/mutiply mapping reads with pairtools dedup with --max-mismatch 1. These filtered reads were subsequently converted to .cool format using cooler (v0.9.1) cload pairs, and then to .mcool format with cooler zoomify, including the --balance option.
Assembly: mm39;hg38
Supplementary files format and content: mcool file contains all valid Micro-C pairs stored in HDF5 format with multiple resolutions
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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) |
| GSE256225 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function [Micro-C] |
| GSE256246 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function. |
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| Relations |
| BioSample |
SAMN40017618 |
| SRA |
SRX23679859 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8091470_B7_Dual_R1.merged.250.mcool |
2.1 Gb |
(ftp)(http) |
MCOOL |
SRA Run Selector |
| Raw data are available in SRA |
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