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

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

PROVIDER: PRJEB48856 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR7365795.fastq.gz	Fastqsanger.gz
	ERR7365796.fastq.gz	Fastqsanger.gz
	ERR7365797.fastq.gz	Fastqsanger.gz
	ERR7365798.fastq.gz	Fastqsanger.gz

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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

Amplicon sequencing of genomically-encoded guide RNA sequences for the Brunello CRISPR library in eHap cells

Project description:eHap CRISPR-Cas9 mutagenized cells with no selection serves as a control. RPS6p antibody staining on the mutagenized eHap cells was used in a flow-sorting approach to isolate RPS6p high and low populations respectively. The genomic DNA from these different samples was isolated, subjected to PCR to amplify the genomically-encoded guide RNA sequences, the product(s) of which was then sequenced on a Hi-Seq X Ten instrument by Novogene.

2020-04-23 | E-MTAB-8926 | 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

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL

Project description:Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL

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

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

| PRJEB73612 | ENA

Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL

Project description:With the near eradication of poliovirus due to global vaccination campaigns, attention has shifted to other enteroviruses that can cause polio-like paralysis syndrome (now termed acute flaccid myelitis (AFM)). In particular, enterovirus D68 (EV-D68) is believed to be the main driver of epidemic outbreaks of AFM in recent years, yet not much is known about EV-D68 host interactions. EV-D68 is a respiratory virus but, in rare cases, can spread to the central nervous system to cause severe neuropathogenesis. We use genome-scale CRISPR screens to identify genes important for EV-D68 infection. A549 and U87-MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library (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,expanded, and seeded for the screens. Each screen had over 500x genome coverage. The cells were infected with EV-D68 IL (BEI USA/2014/18952) (MOI 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.

2025-02-25 | E-MTAB-14765 | biostudies-arrayexpress

Amplicon sequencing of genomically-encoded guide RNA sequences for the Brunello CRISPR library in eHap cells

Project description:Amplicon sequencing of genomically-encoded guide RNA sequences for the Brunello CRISPR library in eHap cells

| PRJEB37745 | ENA

ChIP-seq analyses with eHAP cells

Project description:ChIP-seq analyses with eHAP cells for TFDP1 and histone modifications

2022-05-01 | GSE144451 | GEO

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

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

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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

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