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Sample GSM8057072

Query DataSets for GSM8057072

Status

Public on Jul 26, 2024

Title

S14_SLX-17131.NEBNext16

Sample type

SRA

Source name

Chinese Ovary cells (CHO-K1)

Organism

Cricetulus griseus

Characteristics

cell line: Chinese Ovary cells (CHO-K1)
treatment: Infected_Untreated (UT)_Sorted (S)_Round 1 (R1)_Bright GFP (B)_x2

Treatment protocol

Ttransduced cells were split in two subpopulations, one treated with 2-deoxy-D-glucose (2DG) to induce ER stress and the other untreated. Approximately 6.6 × 107 Mab2C1-stained cells were subjected to FACS. In the screen performed in the presence of ER stress < 2% of cells that had a XBP1s::mCherry+/BiP::sfGFP– phenotype were selected for analysis. For the screen performed in basal conditions < 2% of total sorted cells that had a XBP1s::mCherry-/BiP::sfGFP+ phenotype were selected for analysis. Rounds of enrichment were carried on by sorting, cellular recovering/expansion and sorting. Equal number of transduced cells, untreated or 2DG treated, were passed without sorting as a control group in each round of sorting.

Growth protocol

Approximately 2.1 × 108 dual UPR reporter (ATF6⍺/IRE1⍺, XC45-6S) CHO-K1 Cas9 stable cells were infected at a multiplicity of infection (MOI) of 0.3 to favour infection with a single viral particle per cell. Two days after infection, transduced cells were selected with 8 µg/ml puromycin for 14 days.

Extracted molecule

genomic DNA

Extraction protocol

To prepare samples for deep sequencing, genomic DNA from enriched and sorted populations as well unsorted cells (to represent the entire library) was extracted from ~3.6× 107 and ~1-3× 106 and, respectively, by incubation in proteinase K solution [100 mM Tris-HCl pH 8.5, 5 mM EDTA, 200 mM NaCl, 0.25% (w/v) SDS, 0.2 mg/ml Proteinase K] overnight at 50°C.
Integrated sgRNA sequences were amplified by nested PCR and the adaptors for Illumina sequencing (HiSeq4000) were introduced at the final amplification round. After quantification the products were subjected to NGS sequencing using custom primer and the illumina indexing primer with single-end reads of 50 bp on a hiseq 4000. The sequences were processed and guide counts, gene rankings and statistics were generated using MAGECK software (Li et al., 2014).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

pKLV_NEBNXT09_1767
Single Read, 50bp

Data processing

A comprehensive quality control was performd by using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) computational software. This involved quantification of Total HiSeq read counts, percentage of mapped reads to the CHO library, the number of sgRNA in the library, the number of sgRNA with zero read counts, the GiniIndex of the read count distribution and the frequency distribution of sgRNA in each sample.
Generation of a total read counts table and normalized read count table using MAGECK software (Li et al., 2014)
Identification of positively and negatively selected sgRNA and generation of the corresponding gene list by comparing single samples using MAGeCK tool.
Assembly: Chinese Hamster Ovary (CHO) CRISPR library
Supplementary files format and content: text files and fastq data

Submission date

Jan 31, 2024

Last update date

Jul 26, 2024

Contact name

Adriana Ordonez

E-mail(s)

[email protected]

Organization name

Cambridge Institute for medical Research

Department

Biochemistry