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Sample GSM4803158

Query DataSets for GSM4803158

Status

Public on Jun 02, 2021

Title

S7_SLX-8111.DNAA001_merged

Sample type

SRA

Source name

Cell line (CHO-K1)_ DNA

Organism

Cricetulus griseus

Characteristics

reagent label: DNAA001
treatment: Infected with Lib2 (L2)_Untreated (UT) and Unsorted (US) (duplicate)
cell line: CHO-K1

Growth protocol

2.1×10^8 CHO-K1 cells expressing the polymerogenic α1ATH334D mutant under a tetracycline inducible promoter Tet-on were initially infected with the Library 1 (L1) at a multiplicity of infection (MOI) of 0.3 and selected with 8 µg/ml puromycin for 7 days. Expression of α1AT was induced with 10 ng/ml doxycycline for 24 hrs. Afterwards, the cells were fixed and permeabilised for intracellular staining of α1AT polymers using the polymer-specific monoclonal antibody 2C1 (Mab2C1). Approximately 6.6×10^7 Mab2C1-stained fixed cells were subjected to FACS and collected in 3 bins according to their fluorescence intensity (Mab2C1): ‘brightest' (B), ‘medium' (M) , and ‘dull' (D). Rounds of enrichment were carried on by extracting the genomic DNA of the ‘brightest’-binned fixed cells and recovering by PCR a 220bp fragment containing the sgRNA-bearing region. The amplicon was ligated into the parental lentiviral backbone to generate derivative enriched libraries (called Lib1 [L1] and Lib2 [L2]) that were used to perform two successive cycles of infection of ~2×10^7 parental CHO-K1 Tet-on_α1ATH334D_Cas9 cells. In each round an equal number of infected, untreated (UT) cells (no doxycycline) or uninfected, doxycycline-treated (T) cells were passed without sorting as a control group.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA from fixed, enriched, and sorted populations as well as fixed, unsorted libraries was extracted from ~1-3×106 and ~3.6×107 cells respectively, by incubation in proteinase K solution [100 mM Tris-HCl pH 8.5, 5 mM EDTA, 200 mM NaCl, 0.25% SDS, 0.2 mg/ml Proteinase K] overnight at 50°C. To reverse formaldehyde crosslinks, samples were supplemented with 500 mM NaCl and incubated at 65°C for 16 hrs.
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 HiSeq 4000

Description

Hi Seq 400, Single Read, 50bp

Data processing

Library strategy: CRISPR screen
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.
Genome_build: Chinese Hamster Ovary (CHO) CRISPR library. File used sgRNA sequence is available on the series record.
Supplementary_files_format_and_content: CHO_SLX_8111_190218_counts.txt: raw HiSeq read counts
Supplementary_files_format_and_content: CHO_SLX_8111_190218_counts_normalized.txt: normalized HiSeq read counts
Supplementary_files_format_and_content: S8_S9vsS1_S2.gene_summary.txt: Rank of genes enriched in cells infected with the “derivative enriched Lib2” doxycycline-treated and sorted, relative to cells infected with the “unenriched Lib0” untreated and unsorted

Submission date

Sep 25, 2020

Last update date

Jun 02, 2021

Contact name

Adriana Ordonez

E-mail(s)

aog23@cam.ac.uk, hadrianusca@hotmail.com

Phone

07870368683

Organization name

University of Cambridge