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Sample GSM5678515

Query DataSets for GSM5678515

Status

Public on Aug 29, 2023

Title

cells_R7

Sample type

SRA

Source name

mouse melanoma cell line

Organism

Mus musculus

Characteristics

cell line: HCmel12
pertubation/treatment: CRISPR_Knockout_Kinome_Brie_Library
sample origin: in vitro cells_edited for 49 days

Treatment protocol

HCmel12-Cas9 melanoma cells were transduced at a MOI < 0.1 with lentivirus produced from a kinome-wide lentiviral mouse CRISPR knockout guide library containing 2,852 sgRNAs (Brie, Addgene # 75316) with at least 1000-fold representation (cells per construct) in each infection replicate. Spin-infected ~30 million HCmel12_Cas9 cells with the kinome CRISPR lentivirus library at MOI =0.1 in multiple 6-well plates at Day 0, and transferred the cells into T75 flasks at Day 2. Blasticidin selection (10 µg/ml) started at Day 2, and cells were harvested at Day 7. Puromycin selection stated at day 2 and lasted the entire experiment. On day 14 tumor cells were harvested and used for in vivo screening experiment. In parallel to in vivo screening experiment, cells were kept in culture and continued to edit for 21, 28, 35, 42, 49 and 52 days, the entire duration in parallel to in vivo editing. In vitro edited cells were palleted and used for downstream gDNA extraction and library preparations. For in vivo screening, 3x106 of library-transduced HCmel12-Cas9 cells were injected into the right flank or lateral tail vein of the mice. Tumor growth and signs of sickness was monitored over time. Mice were euthanized after 4 weeks and tumors, lungs, lymph node and liver were evaluated for metastasis presence, lesions dissected for gDNA isolation, amplification of the sgRNA region, and sequencing.

Growth protocol

Mouse melanoma cell line HCmel12 (gift from Prof. Thomas Tueting, Magdeburg, Germany) were grown in ‘complete’ RPMI-1640 medium (Gibson Bioscience) supplemented with 10%FBS (Gibco™ 10437028), 10mM non-essential amino acids, 1mM HEPES (all from Life Technologies), 20 µM 2-mercaptoethanol (Sigma), 100 IU per ml penicillin and 100 µg/ml-1 streptomycin (Invitrogen). Cell lines were routinely tested for mycoplasma contamination using Lonza MycoAlert Kit (Cat. # LT07-318).

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA from cells (edited for 10, 14, 21, 28, 35, 42, 49 and 52 days) and mouse tissue (primary tumor, lung, liver, lymph node), was extracted using Qiagen kit for DNeasy Blood & Tissue Kit (69506,Qiagen).
The procedure of amplifying pooled sgRNA library was based on the protocol described previously (Doench et al., 2016). Sequencing libraries were constructed as previously described (Doench et al., 2016) and sequenced on an Illumina HiSeq2500 Instrument.
sgRNA enrichment sequencing

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

gDNA

Data processing

data processing steps were performed as described previously (Doench et al., 2016). CRISPR–Cas9 screens were analyzed using software packages MAGeCK and MAGeCKFlute. MAGeCK allows to analyze CRISPR/Cas9 screen data, and performs quality control, read count generation and normalization, calculated beta score to evaluate gene selection performance. MAGeCK’s robust rank aggregation (RRA) method was used to identify significantly enriched or depleted CRISPR-screen hits in cells and tumors. MAGeCKFlute R package was used for data analysis and visualization.
For RNAseq data analysis the tximeta R package (Love et al. 2020) was used to import transcript-level quantification data quantified with Salmon package (Patro et al. 2017). Reads were mapped to mouse reference - Mus_musculus.GRCm38.97 and DESeq2 package was applied to generate normalized data.
Genome_build: Mus_musculus.GRCm38.97

Submission date

Nov 08, 2021

Last update date

Aug 29, 2023

Contact name

Meri Rogava

E-mail(s)

mr4020@cumc.columbia.edu

Organization name

Columbia University

Department

Hematology Oncology