|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Aug 29, 2023 |
Title |
cells_R1 |
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_editied for 10 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
|
Lab |
Izar Lab
|
Street address |
650 W 168th St, New York, NY 10032
|
City |
New York |
State/province |
New York |
ZIP/Postal code |
10032 |
Country |
USA |
|
|
Platform ID |
GPL17021 |
Series (1) |
GSE188391 |
A genetic-metabolic axis for metastatic liver organotropism |
|
Relations |
BioSample |
SAMN22980858 |
SRA |
SRX13046640 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|