Comparison of CWC27 knockdown in HEK293T cells compared to scrambled shRNA control

HEK293T cells transduced with shRNA from MISSION library TRCN0000000139 using lentiviral delivery system. HEK293T cells transduced with scrambled shRNA, gifted from Dr. Mauricio Reginato. Sequence is 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′. Tara L Davis, S. RaElle Jackson, Beth Adams, Anh Trinh, Peter Naranjo, and Angie Giang performed primary experimental contributions to cell lines, RNA/cDNA preparation, and validation of results, all Drexel University College of Medicine, Philadelphia, PA 19102. Hetty Rodriguez and John Tobias performed Bioanalyzer and microarray expreriments, and initial data processing. Affiliation: Molecular Profiling Facility and Genomic Analysis Core Bioinformatics Group, University of Pennsylvania. Human CWC27 (aliases: NY-CO-10, Serologically Defined Colon Cancer Antigen 10 or SDCCAG-10) is a cyclophilin, an enzyme that interconverts cis and trans isomers of proline. The CWC27 gene, in addition to the cyclophilin domain, encodes for two low complexity regions and a coiled coil region. CWC27 associates with the human spliceosome, the complex and dynamic machinery that removes intronic sequence from pre-messenger RNA (pre-mRNA). Nothing is known about the function of CWC27 in the nucleus. To understand the function of CWC27, we knocked down CWC27 in human cells. We characterized a set of alternative splicing and transcriptional events that are CWC27-responsive. We used these splicing and transcriptional bioassays to show that CWC27-responsive events are largely specific, even within the cyclophilin family. 3 replicates of CWC27 knockdown cell lines (HEK293T cells, stably integrated, selected using puro, MISSION shRNA TRCN0000000139: 5'-CCGGACGGGAACTCTTAGCAGCAAACTCGAGTTTGCTGCTAAGAGTTCCCGTTTTTT-3'). 3 replicates of SCR control cell lines (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