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| Status |
Public on Jun 15, 2020 |
| Title |
Nascent RNA-seq of human AID-NeonGreen-Tpr tagged DLD-1 cells after loss of Tpr nucleoporin |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
We used SLAM-seq approach to define a contribution of RNA synthesis into RNA abundance changes after Tpr loss. Basket nucleoporin Tpr was AID-tagged and depleted through Auxin Induced Degradation system. Loss of Tpr led to rapid changes in rates of RNA synthesis. The majority of transcripts were downregulated. Analysis of RNA-seq and SLAM-seq indicated that the changes in RNA abundance of most of Tpr-dependent up- and downregulated genes resulted from increased or decreased rates of RNA synthesis, respectively.
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| Overall design |
Thiol (SH)-Linked Alkylation for the Metabolic (SLAM) sequencing of nascent RNAs was performed with 4-Thiouridine (S4U) incorporation using Anabolic (061.24) Kinetics Module kit purchased from Lexogen company. Briefly, before labelling of nascent RNA, each well of DLD-1 AID-Tpr cells from the 6-well plate was replaced with fresh media or media containing 1 mM auxin for 2 hours. For chasing, cells were treated with media containing 300 µM S4U or 300 µM S4U supplemented with 1 mM auxin for 0 min, 30 min, 60 min, or 2 hours. Total RNAs were processed to allow conversion of S4U to cytosine, sequenced, and the T>C reads were used to identify genes that were up- and down-regulated in response to Tpr loss.
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| Contributor(s) |
Aksenova V, Esnault C, Arnaoutov A, Dasso M |
| Citation(s) |
32917881 |
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| Submission date |
May 05, 2020 |
| Last update date |
Sep 16, 2020 |
| Contact name |
Alexei Arnaoutov |
| E-mail(s) |
arnaouta@mail.nih.gov
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| Organization name |
NICHD
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| Department |
PCRM
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| Street address |
49 Convent Dr, Bldg 49 Room 5A10
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (24)
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| GSM4516380 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR_replicate 1 |
| GSM4516381 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR_replicate 2 |
| GSM4516382 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR_replicate 3 |
| GSM4516383 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h_replicate 1 |
| GSM4516384 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h_replicate 2 |
| GSM4516385 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h_replicate 3 |
| GSM4516386 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 30 min_replicate 1 |
| GSM4516387 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 30 min_replicate 2 |
| GSM4516388 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 30 min_replicate 3 |
| GSM4516389 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 30 min_replicate 1 |
| GSM4516390 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 30 min_replicate 2 |
| GSM4516391 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 30 min_replicate 3 |
| GSM4516392 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 1h_replicate 1 |
| GSM4516393 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 1h_replicate 2 |
| GSM4516394 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 1h_replicate 3 |
| GSM4516395 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 1h_replicate 1 |
| GSM4516396 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 1h_replicate 2 |
| GSM4516397 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 1h_replicate 3 |
| GSM4516398 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 2h_replicate 1 |
| GSM4516399 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 2h_replicate 2 |
| GSM4516400 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, S4U 2h_replicate 3 |
| GSM4516401 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 2h_replicate 1 |
| GSM4516402 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 2h_replicate 2 |
| GSM4516403 |
DLD-1 cells with endogenously modified AID-NeonGreen-TPR, auxin treated for 2h, S4U 2h_replicate 3 |
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| Relations |
| BioProject |
PRJNA630447 |
| SRA |
SRP260092 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE149890_RAW.tar |
9.2 Mb |
(http)(custom) |
TAR (of CSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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