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Project description:Molecular profiling of small-molecules offers invaluable insights on compound functionality and allows for hypothesis generation of targets. However, current profiling methods are either limited in the number of measurable parameters or throughput. Here, we developed a multiplexed, unbiased framework that by linking genetic to drug-induced changes in nearly a thousand metabolites allows for high-throughput functional annotation of compound libraries in Escherichia Coli. First, we generated a reference map of metabolic changes from (CRISPR) interference with 352 genes in all major essential biological processes. Next, based on the comparison of essential gene knockdown metabolic profiles with 1342 drug-induced metabolic changes we demonstrated the ability to make de novo predictions of compound functionality and revealed drugs interfering with unconventional antibacterial targets. The same framework that combines dynamic gene silencing with metabolomics we implemented in E. coli can be adapted and applied as a general strategy for comprehensive high-throughput analysis of compound functionality, from bacteria to human cells.
2021-12-16 | PXD024133 | Pride
Project description:MicroRNAs (miRNA) are ~21 nucleotide long, small endogenous non-coding RNAs that functioning in regulation of gene expression found in many eukaryotes. In this study, small RNA libraries of opium poppy from four different tissues (leaf, root, capsule, stem) were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. A total of 27 opium poppy miRNAs which have roles in regulation of alkaloid biosynthesis were identified in this study. A six chip study using miRNA isolated from four separate tissues (capsule, leaf, stem, root). small RNA libraries of opium poppy tissues were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. Furthermore, the novel opium poppy miRNAs were also confirmed by a direct small RNA cloning strategy. The microarray platform were performed to measure and analyze the mirnome of the different opium poppy tissues.
2014-12-15 | E-GEOD-62716 | biostudies-arrayexpress
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare GBM transcriptome profiling (RNA-seq) after shRNA based knockdown of PRKAB1 and to compare gene expression by optimal high-throughput data analysis
2018-06-27 | GSE82183 | GEO
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
2024-03-05 | GSE260830 | GEO
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
2024-03-06 | GSE260829 | GEO
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
2024-03-06 | GSE260828 | GEO
Project description:Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.
2024-03-06 | GSE260827 | GEO
Project description:High throughput screening comparing gene expression in human wildtype and patient (TREX1-mutated) fibroblasts, Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis
2021-05-03 | GSE169244 | GEO
Project description:Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types—comprising 12 genetic perturbations in five cell lines—with 1,440 compound-dose combinations, generating 86,400 chemical-genetic measurements.
2020-10-01 | GSE155855 | GEO
Project description:Purpose: To explore the changes of genes expression in different time points during mouse wound healing. Two wounds tissue from one animal were pooled, three animals were used per time point. The wounds tissue was collected on day 0, 1, 3, 5, 7 after cutting. Skin was cleaned of muscle and fat tissue; total RNA was extracted using the RNeasy mini kits (Qiagen), purified using Direct-zol RNA MiniPrep kit (Zymo Research). Next-generation libraries were prepared using the VAHTS TM mRNA-seq V2 Library Prep Kit for Illumina (Vazyme, #NR601). RNA-seq libraries were run on an Illumina HiSeq X-Ten next-generation sequencer. Analysis of RNA-seq data was done using the DESeq package in R.
2020-06-05 | GSE128193 | GEO