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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Amplicon DNA-sequencing of time series of pooled-CRISPR knock-out screens of Osimertinib and DMSO-treated cell lines

ABSTRACT: This project is a drug-gene interaction CRISPR screen to compare a 3-guide and a 6-guide library.

INSTRUMENT(S): NA, Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER:

PROVIDER: E-MTAB-13979 | biostudies-arrayexpress |

SECONDARY ACCESSION(S): ERP159128

REPOSITORIES: biostudies-arrayexpress

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Amplicon DNA-sequencing of time series of pooled-CRISPR knock-out screens of Osimertinib and DMSO-treated cell lines using a dual-guide CRISPR library

Project description:This project is a drug-gene interaction dual-guide CRISPR screen using a 3-guide pairs per gene library design.

2025-02-12 | E-MTAB-13980 | biostudies-arrayexpress

Amplicon DNA-sequencing of time series of pooled-CRISPR knock-out screens of colon cancer cell lines

Project description:This project aims to compare gRNA design algorithms using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.

2025-02-12 | E-MTAB-13976 | biostudies-arrayexpress

Amplicon DNA-sequencing of time series of pooled-CRISPR knock-out screens of the HT29 colon cancer cell line

Project description:This project also aims to compare gRNA design algorithms using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.

2025-02-12 | E-MTAB-13977 | biostudies-arrayexpress

Amplicon DNA-sequencing of time series of pooled-CRISPR knock-out screens of cell lines using a dual-guide CRISPR library

Project description:This project aims to compare gRNA design algorithms and single- versus dual-targeting using a benchmark CRISPR-knockout library composed of gRNAs targeting essential and non-essential genes.

2025-02-12 | E-MTAB-13981 | biostudies-arrayexpress

shRNA Sensor Analysis of 5' U converted shRNAs reveals 3' target bias exists in addition to 5' guide bias

Project description:shRNAs selected with the shERWOOD algorithm were converted to have a U at the 5' end of their guide. When endogenous 1U shRNAs were compared to artificial shRNA via the sensor algorithm, the endogenous shRNAs were found to be more efficacious. Purpose: Structural studies have hinted that the 5' end of shRNA guides is engulfed in the RISC complex. It has also been reported that shRNAs with a 5' U are more efficacious than those with other 5' caps. We wished to determine whether replacement of shRNA guide 5' nucleotides with a U, regardless of the corresponding target base, would increase their efficacy. Method: For each gene in the "druggable genome" 10 shRNAs were selected with the shERWOOD algorithm. In each case the score was assessed as if the guide had a 5' U. Sensor constructs were designed pairing 1U-guide shRNAs with their endogenous target. shRNAs were assessed for efficacy via the shRNA sensor assay (Fellmann et al. Mol Cell 2011). Results: shRNAs with artificial 5' Us were found to be less efficacious than those with an endogenous 5' U,

2014-11-24 | E-GEOD-62186 | biostudies-arrayexpress

Sequence database choice significantly affects taxonomic and functional metaproteomic results in gut microbiota studies

Project description:Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics (the study of the whole protein complement of a microbial community) can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Here we present a systematic investigation of variables concerning database construction and annotation, and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. Taxonomic and functional results were revealed to be strongly database-dependent, especially when dealing with mouse samples. As a striking example, in mouse the Firmicutes/Bacteroidetes ratio varied up to 10-fold depending on the database used. Finally, we provide recommendations regarding metagenomic sequence processing aimed at maximizing gut metaproteome characterization, and contribute to identify an optimized pipeline for metaproteomic data analysis.

2016-10-10 | PXD004039 | Pride

Amplicon Sequencing of gRNAs from Druggable Genome CRISPR mutagenized Huh7.5.1 cells after live/dead selection with DENV2

Project description:Huh7.5.1 cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, Huh7.5.1 cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the druggable genome library at MOI 0.3. Cells were then selected with puromycin, expanded, and then pooled together and cryofrozen in aliquots. Cells were thawed constituting over 1000× genome coverage worth of mutagenized library. The cells were infecting with DENV2_429557 and DENV-2_16681_Hap1-adapted at MOI of 0.1. Virus-resistant colonies were harvested. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.

2024-12-29 | E-MTAB-14693 | biostudies-arrayexpress

Changes in intestinal permeability and gut microbiota following diet-induced weight loss in patients with metabolic dysfunction-associated steatohepatitis and liver fibrosis

Project description:We explore whether a low-energy diet intervention for Metabolic dysfunction-associated steatohepatitis (MASH) improves liver disease by means of modulating the gut microbiome. 16 individuals were given a low-energy diet (880 kcal, consisting of bars, soups, and shakes) for 12 weeks, followed by a stepped re-introduction to whole for an additional 12 weeks. Stool samples were obtained at 0, 12, and 24 weeks for microbiome analysis. Fecal microbiome were measured using 16S rRNA gene sequencing. Positive control (Zymo DNA standard D6305) and negative control (PBS extraction) were included in the sequencing. We found that low-energy diet improved MASH disease without lasting alterations to the gut microbiome.

2024-12-31 | E-MTAB-14061 | biostudies-arrayexpress

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized HT29-DKO cells after live/dead selection with PeV-A1 and PeV-A2

Project description:HT29-DKO cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million HT29-DKO cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. The cells were infecting with PeV-A1 or PeV-A2 at an MOI of 0.1. Virus-resistant colonies were harvested. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.

2024-03-16 | E-MTAB-13894 | biostudies-arrayexpress

High-throughput CRISPR screen to identify factors involved in DNA methylation maintenance

Project description:Mouse embryonic stem cells have been modified with a DNA methylation reporter based on the Sprn promoter system published in Stelzer et al. (Cell, 2015). This reporter allows selection by Hygromycin as well as mScarlet expression. The cells were infected with a Lentiviral genome-wide CRISPR/Cas library resulting in the upregulation of Hygromycin/mScarlet when cells lose global DNA methylation.

2022-01-01 | E-MTAB-9126 | biostudies-arrayexpress

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