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Sample GSM8242602

Query DataSets for GSM8242602

Status

Public on May 01, 2024

Title

Top1

Sample type

SRA

Source name

HFF-1

Organism

Homo sapiens

Characteristics

cell line: HFF-1
cell type: fibroblasts
treatment: Collected from the top surface of transwell after the first round of selection

Treatment protocol

Cells were transduced with the Human Genome-Scale CRISPR Knock-Out (GeCKO) lentiviral pooled library (GeCKO v2, library A, cat. 1000000048, Addgene), and cultured either in standard flasks or in transwell devices.

Growth protocol

HFF were maintained in Dulbecco's modified Eagle Medium (DMEM, cat. 11965, ThermoScientific) supplemented with 15% fetal bovine serum (FBS, ThermoScientific) at 37°C and 5% CO2. Cells were maintained on culture-ware (including transwells) coated with Matrigel growth factor reduced (MGFR, 50 μg/mL overnight at 4°C and 1 h at 37°C, cat. 354230, Corning) and passaged by 0.25% trypsin digestion (ThermoScientific) before reaching confluency.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was extracted using Quick-gDNA MidiPrep (Zymo Research) following manufacturer's instructions
Genomic DNA amplification for Next Generation Sequencing analysis was performed according to Joung et al. (10.1038/nprot.2017.016)
Library preparation for Next Generation Sequencing analysis was performed according to Joung et al. (10.1038/nprot.2017.016)

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

Illumina binary base call (BCL) files were converted into fastq file format by Illumina bcl2fastq software (v. 2.17).
Paired reads were merged using Pear (v. 0.9.11), and trimmed using cutadapt (v. 2.8).
The trimmed reads were mapped to the human GeCKOv2 library by Bowtie (v. 1.2.3).
The sgRNA count matrix was obtained from Bowtie output sam files.
Data from each sample were merged into a single table of unfiltered counts with R software (v. 4.2.2).
Assembly: Human Genome-Scale CRISPR Knock-Out (GeCKO) lentiviral pooled library (GeCKO v2, library A, cat. 1000000048, Addgene)
Supplementary files format and content: Tab delimited file containing the unfiltered counts from Bowtie alignment

Submission date

Apr 30, 2024

Last update date

May 01, 2024

Contact name

Camilla Luni

Organization name

University of Bologna

Department

DICAM

Street address

Via Terracini 28