Project description:<p>The Prostate Cancer Medically Optimized Genome-Enhanced Therapy (PROMOTE) study uses genetic clues in castration-resistant prostate cancer that may identify an individualized treatment approach for men with the disease.</p> <p>Understanding the molecular biology behind castration-resistant prostate cancer has led to more treatment options, but there are still no definite conclusions about which specific drug best treats patients - maximum suppression of cancer growth while minimizing side effects.</p> <p>The PROMOTE study explores the genetic characteristics of each tumor to predict these treatment paradigms for the future, resulting in more effective and less toxic options for patients.</p> <p>Our long-term goal is to improve treatments for men with advanced prostate cancer by using genomic sequencing to increase life span and quality of life. We also will uncover novel vulnerable targets in the cancer genome that may provide new drug therapies.</p> <p><i><b>PARTICIPATION</b></i></p> <p>Eligible participants are men:</p> <p> <ul> <li>With castration-resistant prostate cancer or prostate cancer not responding to hormone treatments</li> <li>About to begin abiraterone acetate therapy</li> <li>Agreeable to undergoing two tumor biopsies</li> </ul> </p> <p>During the study, participants travel to Mayo Clinic for an initial biopsy (before beginning abiraterone acetate) and a second biopsy approximately three months later. The cell tissue collected is analyzed to identify gene alterations in the tumor that could eventually be targeted with treatments. Tissue is preserved for future research.</p> <p>Participants can continue to be treated by their local cancer care team during this period and beyond. In addition, the Mayo team carefully monitors participants' cancer via follow-up studies and the genetic signature of tumors that were biopsied so that patients may benefit from future treatments.</p>
Project description:Linear amplification of RNA by T7 bacteriophage polymerase is widely used in molecular biology. We performed 5’RACE-Seq to identify T7 promoter variants with enhanced transcriptional activity that generate up to five-fold higher RNA output in large scale synthesis reactions. In single-cell RNA-Sequencing, optimized T7 promoters facilitate library preparation, and substantially increase library complexity and the number of expressed genes detected per cell, highlighting a particular value for bioanalytical applications
Project description:We optimzed ATAC-seq library preparation for use with Drosophila melanogaster. The protocol addresses factors specific to fruit flies, such as the insect exoskeleton and smaller genome size. The optimized protocol provides guidelines for sample input, nuclei isolation, and enzymatic reaction times. The data included here were generated using our optimized library preparation workflow.
Project description:CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a system (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.