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| Status |
Public on Mar 07, 2024 |
| Title |
JM8.N4 mESC, clone A ∆ZFP143/∆CTCF 3h, PRO-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: Drosophila melanogaster
spike-in cell_line: S2
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 |
nuclear RNA |
| Extraction protocol |
Cells were permeabilized using a combination of 0.1% (v/v) Igepal CA-630 and 0.05% (v/v) Tween-20. Subsequently, biotin-11 dNTPs were incorporated into the 3’ end of nascent RNA transcripts in a biotin run-on reaction. The run-on was stopped and total RNA was extracted using the Norgen Biotek Total RNA Purification Kit. The RNA was chemically fragmented and biotin-enriched using streptavidin beads.
The 3’ and 5’ adapters were ligated to the extracted RNA. The adapter-ligated RNA was converted to a cDNA library by reverse transcription using the SSIV RTase. Libraries were indexed by PCR using the NEBNext Ultra II Q5 Master Mix and custom primers from IDT. Libraries were purified using ProNex (Promega) beads. Purified libraries were quantified by qPCR and pooled in a 1:1 ratio. Following qPCR quantification, libraries were submitted for sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
proseq_a_dual_r2
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| Data processing |
*library strategy: PRO-seq
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 dm6 genomes using bowtie2.
Spike-in and primary reads were independently deduplicated using UMI-tools and sorted using samtools 1.3.1 -n. These were converted to bedGraph format using a custom script (bowtie2stdBedGraph.pl), and subsequently converted to bigwig format.
Assembly: mm39; dm6
Supplementary files format and content: bedGraph file has data based on all uniquely aligning non-duplicate reads
Library strategy: PRO-seq
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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) |
| GSE256227 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function [PRO-seq] |
| GSE256246 |
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function. |
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| Relations |
| BioSample |
SAMN40017914 |
| SRA |
SRX23679687 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8091558_Hansen001_PR1138_A_dualDep_rep2_mm39_dedup_3pr_forward.bedGraph.gz |
66.2 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM8091558_Hansen001_PR1138_A_dualDep_rep2_mm39_dedup_3pr_reverse.bedGraph.gz |
64.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
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