Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Genome-wide CRISPR screening combined with ATAC-see assay

ABSTRACT: Cas9 expressing eHAP cells were transduced with human sgRNA lentivirus library (Doench, 2016 Nature biotech, Addgene, #73178). The cells were selected with 1 µg/ml of puromycin for 2 days, and then used for ATAC-see at 5 and 7 days post-transduction, respectively. Cells in the G2 phase were specifically gated to sort th 5% of the cells with the highest and lowest ATAC-see signal intensity(“high” or “low” population) . The sgRNA enrichment in each population was analysed by high-throughput sequencing and computationally compared by using the MAGeCK program (Li et al., Genome Biology 2014).

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE144450 | GEO | 2022/05/01

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized U87MG or eHAP cells after live/dead selection with Reovirus Type 3 Dearing Strain.

Project description:eHAP or U87MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million eHAP or U87MG 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. Twenty T175 flasks were used for the U87MG-based screen, while twelve T175 flasks were used for the eHAP-based screen. The cells were allowed to recover for 48 hours before infecting with ReoT3D at an MOI of 0.1. Obvious CPE was observed within 72 hours. eHAP-resistant colonies were harvested two weeks later, while U87MG-resistant colonies were harvested six weeks later. 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.

2022-09-10 | E-MTAB-11158 | biostudies-arrayexpress

RNA-seq analyses with TFDP1 knockout eHAP cells

Project description:Cas9 expressing eHAP cells were transfected with sgRNA against TFDP1. 5 days after the transfection, polyA RNA was analyzed by RNA-seq

2022-05-01 | GSE144453 | GEO

RNA-sequencing of SKBR3 cells with and without NTN4 overexpression

Project description:Low NTN4 expression is correlated with breast cancer proliferation and metastasis, while the molecular mechanism of NTN4 has not been fully explored. Here, the breast cancer cells SKBR3 stably expressing dCas9-VP64 and MS-P65-HSG1 were generated by transducing a lentiviral plasmid containing lenti-dCAS-VP64-Blast (Addgene plasmid # 61425) and lenti-EF1a-MS2–p65–HSF1-2A-Hygro-WPRE (Addgene plasmid # 89308), followed by selection and maintained using 10 μg/mL of blasticidin and 200 μg/mL of hygromycin. SKBR3-dCas9-VP64-MPH cells were further transduced with a doxycycline (Dox)-induced expressing sgRNA targeting NTN4 promoter. The successfully transfected cells were selected and maintained with puromycin at a concentration of 0.5 μg/mL for SKBR3-dCas9-VP64-MPH. SKBR3-dCas9-VP64-MPH-AC-sgRNA cells were treated with or without 50 ng/μL Dox (three biological repeats for both groups) to induce NTN4 overexpressing. Total RNA was extracted using TRIzol according to the manufacturer’s instructions. The quality of RNA samples was examined by Agilent BioAnalyser 2100. The samples were subjected to RNA-sequencing analysis on the BGISEQ-500 system by Beijing Genomics Institute (BGI, China). The clean-tag reads were aligned to the reference genome and reference genes using HISAT2 and Bowtie2. The matched reads were calculated and then normalized using RSEM software. Differential gene expression was analyzed using DESeq2 and was carried out further to explore the mechanism of NTN4 in breast cancer.

2022-05-27 | GSE197635 | GEO

Gene expression profile of LNCaP prostate cancer cells with exogenous HNF4G expression

Project description:To determine genes regulated by HNF4G, we retrovirally infected LNCaP cells in triplicates to express HNF4G and empty vector as control. cDNA for HNF4G in pDONR201 vector was obtained from Harvard medical school Plasmid database (ID:HsCD00022314) and was cloned into an murine stem cell virus (MSCV)-based retroviral vector with puromycin selection marker (Addgene) using Gateway technology.

2017-11-01 | GSE85244 | GEO

Transcriptome profiling of A549 RB1 wild-type/knockout cell lines

Project description:A549 cells were co-transfected with CRISPR/Cas9 (containing sgRNA and GFP) and HDR donor plasmids (containing RFP and puromycin). The genetic editing cells were selected by puromycin. And then, we performed RNA sequencing for transcription profiling of A549 RB1 RB1 wild-type/knockout cell lines. The raw data were processed with Tophat and cuffdiff for gene expression analysis.

2020-08-25 | GSE132722 | GEO

Genome wide CRISPR screen for CG-806 resistance in OCI-LY3 cells using Y. Kosuke library

Project description:OCI-LY3 cells were infected with CRISPR/Cas9 library, selected for puromycin resistance for integration events and exposed to CG-806 or vehicle, DMSO, at 1 microMolar concentrations. Cells were collected at time 0 (post puromycin selection) and day 7. DNA was extracted and sgRNA barcodes were amplified. PCR library was deep sequenced using highthroughput Illumina platform Novaseq 6000.

2023-05-04 | GSE174766 | GEO

Genome-wide CRISPR knockout screen using GeCKO v2 to identify synthetic lethal interactions with the PARP inhibitor Olaparib

Project description:In this study, a population of knockout cells was generated using the GeCKO v2 sgRNA library. The population of knockout cells was treated with the PARP inhibitor olaparib for 14 days. Over this time, knockout cells that were sensitive to olaparib treatment would die and a population of knockout cells that was resistant to olaparib treatment remained. Cells from olaparib and DMSO conditions and the sgRNA cassette in the knockout cells was amplified and sequenced to identify synthetic lethal or suppressor interactions with olaparib treatment.

2020-08-01 | GSE145743 | GEO

ATAC-seq analyses with knockout eHAP cells for effector genes

Project description:A genome-wide screening identified effector genes of chromatin accessibility. To investigate the functional consequences of the loss of each effector gene, we conducted ATAC-seq analysis with eHAP cells knocked out for each effector gene.

2022-05-01 | GSE144448 | GEO

Single-cell sequencing of JMJD1C Jumonji domain mutated MLL-AF9 Leukemia cells

Project description:We performed single-cell sequencing on mouse MLL-AF9-cas9 leukemia cells 7 days after transduction with sgRNA against Renilla or JMJD1C JmjC domain. We revealed heterogeneity within each population.

2019-04-12 | GSE129662 | GEO

A genetic-metabolic axis for metastatic liver organotropism

Project description:To generate CRISPR/Cas9 knockout screen, we used mouse Kinome CRISPR Knockout Library (Brie) (Addgene # 75316) purchased from addgene. The mouse kinase sgRNA pool includes 2,852 sgRNAs targeting 713 kinase genes and 100 control non-targeting sgRNAs. Cas9 expressing HCmel12 melanoma cells were edited for 14 days, and tumor cells were injected for metastasis screening studies. RNA-seq data: HCmel12 melanoma cells and CRISPR knockout HCmel12 melanoma cells - Pip4k2c KO were treated for 4 hours with insulin. Then cells were lysed in RLT buffer and used to perform mRNA profiling. We performed single-cell RNA-sequencing (scRNA-seq) of eight mice with concurrent liver and lung metastasis. A total of 95,606 cells expressing 19,624 genes were included for downstream analyses. Bulk RNA-sequencing analysis of 15 human melanoma liver metastasis samples from FFPE sample. Tumor areas were macro-dissected and RNA was extracted using Qiagen kit All Prep DNA/RNA FFPE Kit (Catalog Number 80234), concentration was quantified using Qu-bit and stored at-80 °C until use.

2023-08-29 | GSE188391 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data