NGS-based Genome-wide Genetic Screens for RNA Processing Regulators
Recorded On: 02/07/2018
High throughput genetic and chemical screens have been powerful tools to comprehensively identify regulators in specific cellular pathways or drug leads in both industry and academia. However, most screens rely on one or a few functional readouts, even with so-called high content screens. We have developed a robust and fully automatable screen platform that couples with NGS (next-generation sequencing) to monitor the expression of hundred-to-thousand endogenous genes associated with a phenotype without need to purify RNA. The platform is based on annealing a cohort of specific oligonucleotides to specific transcripts and/or their isoforms followed by solid phase selection, RNA-templated oligonucleotide ligation, and PCR amplification using bar-coded primers. We refer this assay strategy as RASL-seq (RNA Annealing, Selection, Ligation coupled with NGS). Pooled samples from up to 1500 reactions in 384 wells can then be sequenced in a single lane of an Illumina sequencing flowcell to obtain quantitative information on targeted transcripts. We have previously identified a gene signature associated with activated androgen receptor (AR) on prostate cancer cells and applied RASL-seq to identify chemicals that can specific inactivate the AR pathway, thus establishing the proof-of-concept for gene signature-based chemical screens, which are equally applicable to both druggable and non-druggable targets. We have also utilized the RASL-seq platform to screen for regulators involved in the regulation of pre-mRNA splicing and alternative polyadenylation in mammalian cells. Coupled with our efforts in technology development, we have developed a bioinformatics pipeline to process the data for quantitative and network analyses. The RASL-seq platform thus offers a general solution to pathway dissection in both genetic and chemical screens.
University of California, San Diego
Name: Hai-Ri Li Affiliation: University of California, San Diego
Position Institution Year(s)
Project Scientist UC, San Diego 2007-Present
Postdoc Fellow UC, San Diego 2002-2006
Postdoc Fellow Sanford-Burnham Medical Research Institute 2000-2001
EDUCATION / TRAINING:
Institution or Location Degree Year(s) Field of Study
China Medical University MS 1986-1989 Endocrinology, Medicine
Jiamushi Medical College, China MD 1977-1983 Medicine
EXPERTISE AND RELEVANT EXPERIENCE
I have been involved in genomics, especially in RNA. I developed two simple RNA-seq methods, one for whole transcriptome profiling and another for 3’ end of RNA profiling. In addition, I developed a target gene profiling method, i.e. RASL-seq (RNA-mediated Annealing, Selection and Ligation, combined with high-throughput sequencing). We applied it to chemical screening in prostate cancer cells. We selected hundred genes in androgen receptor pathway as signatures and observe which chemical compounds affect androgen receptor pathway genes. We sequenced 1536 samples in single lane of Illumina flowcell and found a few promising candidates for prostate cancer therapy. Recently we also applied it to identification of RNA processing regulators and found new previously undescribed RNA processing regulators, which will be helpful in exploring new mechanisms in different diseases.