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| Status |
Public on May 07, 2024 |
| Title |
HEK_CloneZD29_30min_dTAGV1_PRO-seq rep3 |
| Sample type |
SRA |
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| Source name |
HEK_CloneZD29
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK_CloneZD29
genotype: ZNF143 tagged with an inducible degron tag
treatment: 50nM dTAG-V-1 for 30 minutes
molecule subtype: nascent RNA
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| Treatment protocol |
HEK-293T cells were collected after either no treatment or after 30 minutes of 50nm dTAGV-1 treatment
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell permeabilization was performed as previously described (Mahat et al. 2016), with modifications. At the time of harvest, cells were scraped in 10mL ice cold PBS and washed in 5mL buffer W (10mM Tris-HCl pH 7.5, 10mM KCl, 150mM sucrose, 5mM MgCl2, 0.5mM CaCl2, 0.5mM DTT, 0.004U/mL SUPERaseIN RNase inhibitor (Invitrogen), Complete protease inhibitors (Roche)). Cells were permeabilized by incubating with buffer P (10 mM Tris-HCl pH 7.5, KCl 10 mM, 250 mM sucrose , 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40, 0.5 mM DTT, 0.004 units/mL SUPERaseIN RNase inhibitor (Invitrogen), Complete protease inhibitors (Roche)) for 3 minutes on ice. Cells were washed with 10 mL buffer W before being transferred into 1.5mL tubes using wide bore pipette tips. Finally, cells were resuspended in 50μL buffer F (50mM Tris-HCl pH 8, 5mM MgCl2, 0.1mM EDTA, 50% Glycerol, 0.5 mM DTT). Cells were snap frozen in liquid nitrogen and stored at -80°C.
PRO-seq libraries were prepared as previously described (Judd et al. 2020), with modifications. RNA extraction after the run-on reaction was performed with 500μL Trizol LS (Thermo Fisher) followed by 130μL chloroform (Sigma). The equivalent of 1μL of 50μM for each adapter was used. A random eight base unique molecular identifier (UMI) was included at the 5′ end of the adapter ligated to the 3′ end of the nascent RNA. 37°C incubations were performed with rotation with 1.5mL tubes placed in 50mL conical tubes in a hybridization oven. For the reverse transcription reaction, RP1 was used at 100μM and dNTP mix was used at 10mM each. Libraries were amplified by PCR for a total of 8 cycles in 100μL reactions with Phusion polymerase (New England Biolabs). No PAGE purification was performed to ensure that our libraries were not biased against short nascent RNA insertions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
HEK_CloneZD29_30min_dTAGV1_minus_PE1_scaled.bigWig
HEK_CloneZD29_30min_dTAGV1_minus_PE2_scaled.bigWig
HEK_CloneZD29_30min_dTAGV1_plus_PE1_scaled.bigWig
HEK_CloneZD29_30min_dTAGV1_plus_PE2_scaled.bigWig
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| Data processing |
*library strategy: PRO-seq
Adapter trimming: cutadapt v3.5
FASTQ deduplication:https://github.com/guertinlab/fqdedupv1.0.0
FASTQ pairing: fastq_pair v1.0
UMI trimming and reverse complementing: fastX-trimmer and seqtk v1.3
Alignment: bowtie2
File manipulation: samtools v1.9
File manipulation: https://github.com/guertinlab/seqOutBias v1.4.0
Assembly: hg38
Supplementary files format and content: Stranded bigWig counts files
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| Submission date |
May 02, 2024 |
| Last update date |
May 07, 2024 |
| Contact name |
Jinhong Dong |
| E-mail(s) |
jdong@uchc.edu
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| Organization name |
University of Connecticut
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| Department |
Center for Cell Analysis and Modeling
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| Street address |
400 Farmington Ave
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE266491 |
ZNF143 binds DNA and stimulates transcription initiation to activate and repress direct target genes (PRO-seq) |
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| Relations |
| BioSample |
SAMN41178867 |
| SRA |
SRX24438407 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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