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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

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

PROVIDER: PRJEB73612 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR12725690.fastq.gz	Fastqsanger.gz
	ERR12725691.fastq.gz	Fastqsanger.gz
	ERR12725692.fastq.gz	Fastqsanger.gz

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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: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.

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