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Sample GSM8224191

Query DataSets for GSM8224191

Status

Public on Apr 27, 2024

Title

B3Rd4 Infected

Sample type

SRA

Source name

K562

Organism

Homo sapiens

Characteristics

cell line: K562
cell type: Myeloid leukemia lymphoblast
mutagenesis library: Targeted sub-library
treatment: DENV infected via ADE, GFP-

Treatment protocol

Sixty four million (GeCKO v2 library) or 32 million (custom sub-library) mutagenized cells were infected with single-round infectious dengue virus serotype 2 (DENV2-GFP) (Cat # RVP-201, Integral Molecular, Inc) at an MOI of 24 in the presence of 1.25 ug/mL anti-DENV2 mouse antibody DV2-70 (PMID: 20592088) by spinoculation. Two days later, to isolate ADE-resistant cell populations, GFP-negative cells were bulk sorted (Sony SH800 for genome-wide screen, Sony MA9000 for targeted sub-library screen) and allowed to recover and multiply for five days at 37°C prior to re-infection under the above conditions. After three total rounds of infection and bulk sorting, genomic DNA was harvested for deep sequencing.

Growth protocol

K562 cells were maintained in RPMI 1640 supplemented with Gluta-MAX , 7% fetal bovine serum, and 100 U/mL penicillin-streptomycin, at 37°C in 5% CO2

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Cat #51185, Qiagen), and gRNA sequences were amplified and prepared for Illumina sequencing via the NextSeq (genome-wide screen) or MiSeq (targeted screen) platforms.
Genomic DNA was amplified by a two-step PCR to amplify sgRNA sequences (genome-wide screen PCR1: 18 cycles PCR 2: 20 cycles, targeted sub-library screen PCR1: 12 cycles PCR2: 20 cycles). One thousand- or five hundred-fold coverage (for the genome-wide and targeted sub-library screens, respectively) of the sgRNAs were amplified with maximum of 2 ug of genomic DNA per reaction. PCR1 reactions of each sample were pooled and either used directly for PCR2 (genome-wide screen) or first cleaned up with a QIAquick PCR Purification Kit (targeted sub-library screen) before PCR2, which include Illumina barcodes and adaptor sequences. PCR2 products were then pooled, run via gel electrophoresis, and bands were extracted and cleaned up using the QIAquick Gel Extraction Kit. Targeted sub-library samples were then purified by double-sided bead cleanup (AMPureXP, Beckman Coulter). Samples were quantified with a Qubit dsDNA HS Assay and pooled.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Data processing

Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18.54.4 software followed by generation of fastq files using Illumina's bcl2fastq Conversion Software v2.20.
Samples were demultiplexed using FASTX barcode splitter.
Reads were trimmed and aligned to the respective guide libraries using Bowtie v1.0.0, followed by tabulation of read counts using R.
Liu lab's CRISPR analysis tool, Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK), was used to perform differential guide analysis between various groups of interest. PMID: 25476604.
Supplementary files format and content: Tab delimited files with read counts for each guide sequence
Library strategy: CRISPR

Submission date

Apr 23, 2024

Last update date

Apr 27, 2024

Contact name

Laura Belmont

Organization name

Fred Hutchinson Cancer Research Center

Street address

1100 Fairview Ave N

City

Seattle