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| Status |
Public on Oct 01, 2018 |
| Title |
Comparison of ZNF830 knockdown in HEK293T cells to scrambled shRNA control cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
HEK293T cells transduced with two shRNAs from MISSION library (TRCN0000180560 and TRCN0000148198) using lentiviral delivery system. HEK293T cells transduced with scrambled shRNA, gifted from Dr. Mauricio Reginato. Sequence is 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′. Primary contributions to creation of cell lines, preparation of RNA/cDNA for microarray, and validation of results were from Tara L Davis, S. RaElle Jackson, Beth Adams, Anh Trinh, Jessica Kopenhaver, Alyson Hurlock, Angie Giang, and Purva Vaidya, all Drexel University College of Medicine, Philadephia PA, 19102. Hetty Rodriguez and John Tobias, affiliated with the Molecular Profiling Facility and Genomic Analysis Core Bioinformatics Group at the University of Pennsylvania, Philadelphia PA, performed Bioanalyzer and microarray expreriments and initial data processing.
Zinc Finger 830 (ZNF830) is a scaffolding protein, consisting of an N-terminal C2H2 zinc finger motif and a C-terminal domain of unknown function (DUF). ZNF830 associates with the human spliceosome, the complex and dynamic machinery that removes intronic sequence from pre-messenger RNA (pre-mRNA). Although ZNF830 knockdown in mice is lethal due to cell cycle defects, a common phenotype for regulators of alternative splicing, little is known about the targets of ZNF830 transcription and splicing regulation in human cells. To understand the function of ZNF830 in the nucleus, we knocked down ZNF830 in human cells. We characterized a set of alternative splicing and transcriptional events that are ZNF830-responsive. We used these splicing and transcriptional bioassays to show that ZNF830-responsive events are largely specific. The development of a bioassay for ZNF830 function can be used to answer fundamental questions about the role of spliceosomal proteins in regulating splicing and other nuclear functions.
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| Overall design |
3 replicates of ZNF830 knockdown cell lines in HEK293T cells, stably integrated, selected using puro, shRNAs TRCN0000180560 sequence 5'-CCGGGAGAATGCTGACAGCGATGATCTCGAGATCATCGCTGTCAGCATTCTCTTTTTTG-3' and TRCN0000148198 sequence 5'-CCGGGAAACGGATAGAATCTCCATTCTCGAGAATGGAGATTCTATCCGTTTCTTTTTTG-3'. 3 replicates of SCR control cell lines in HEK293T cells, stably integrated, selected using puro, sequence 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′. Total RNA purified from ~1 million cells, cDNA converted using random hexamer primers, quantity measured using NanoDrop, quality quantified using BioAnaylzer). Affymatrix HTA2.0 array
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| Contributor(s) |
Davis TL, Jackson SR, Adams B, Trinh A, Kopenhaver J, Hurlock A, Giang A, Vaidya P, Rodriguez H, Tobias J |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R00 GM094293 |
Functional and structural characterization of spliceosomal cyclophilins |
DREXEL UNIVERSITY |
Tara L Davis |
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| Submission date |
Jul 19, 2018 |
| Last update date |
Oct 29, 2018 |
| Contact name |
Tara Davis |
| E-mail(s) |
Tara.Davis@DrexelMed.edu
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| Organization name |
Drexel University College of Medicine
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Lab 10127
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| Street address |
245 N. 15th St. MS 497
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19102 |
| Country |
USA |
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| Platforms (2) |
| GPL17585 |
[HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [probe set (exon) version] |
| GPL17586 |
[HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [transcript (gene) version] |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA481971 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE117390_RAW.tar |
390.5 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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