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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2022 |
| Title |
v-abl transformed B cells, Chk1i LY2603618, run2, rep2 |
| Sample type |
SRA |
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| Source name |
bone marrow
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| Organism |
Mus musculus |
| Characteristics |
tissue: bone marrow
cell type: v-abl transformed B cells
treatment: Chk1i LY2603618
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| Treatment protocol |
Cells were infected with a tetracycline inducible Cas9 (Addgene 50661), and a single clone was picked for high inducibility and low leakiness. Cells were then infected with a BFP-tagged whole genome mouse gRNA library (Addgene 67988) with an MOI ~1 and then FACS-sorted for BFP+ cells. At least 30 million cells were then treated with 3ug/ml doxycycline (Sigma-Aldrich) for 5 days, refreshing media every other day. Induced cells were then treated with the IC90 dose of drug (as measured by MTT assay (Roche) at 6 days) or DMSO for 6 days, with at least 30 million cells for each treatment to start, refreshing media as necessary. The IC90 doses used were VE821 (Selleckchem): 1.8uM, AZD6738 (Selleckchem): 0.4uM, LY2603618 (Selleckchem): 0.4uM, DMSO: 0.02%.
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| Growth protocol |
v-abl transformed B-cells were grown in DMEM media with 15% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At least 30 million cells were collected in triplicate for each treatment before dox-induction, after dox-induction but before drug treatment, and after 6 days of drug treatment. Cells were lysed and genomic DNA was extracted.
gRNA from extracted genomic DNA was amplified according to (Joung J, et al., Nat Protoc, 2017) using 2.5ug DNA x 96 reactions per treatment, NEBNext High Fidelity Master Mix (2x) (New England Biolabs) and NGS-Lib-Fwd and NGS-Lib-KO-Rev primers. PCR reactions were purified using Zymospin columns (Zymo Research) and cleaned up using AMPureXP beads (Beckman Coulter).
Amplicon-seq of gRNAs harvested from cells.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
20220118_LY2603618_2
gRNA PCR from genomic DNA
labeled: Chk1iLY2603618-2
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| Data processing |
The results were analyzed via the MAGeCK FluteMLE pipeline as previously described (Wang B, et al., Nat Protoc, 2019) using the DMSO samples as the baseline.
Genes with positive z-score and FDR<0.2 identified in the CRISPR screen were analyzed for Gene Ontology (GO) using R studio.
Assembly: Mouse Improved Genome-wide Knockout CRISPR Library v2 developed by Kosuke Yusa was used. A list of gRNAs and genes included can be found on the Addgene listing (67988).
Supplementary files format and content: *.raw_count.xlsx: Processed data files are Excel files of raw gRNA counts generated by the MAGeCK software for all gRNAs represented in the gRNA library used. Each file contains treatments and replicates that were done together in the same experiment.
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| Submission date |
Oct 02, 2022 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Shan Zha |
| E-mail(s) |
sz2296@cumc.columbia.edu
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| Organization name |
Columbia University
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| Street address |
1130 Saint Nicholas Ave.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE212196 |
ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion. |
| GSE214643 |
ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion [CRISPR-Cas9 gRNA screen] |
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| Relations |
| BioSample |
SAMN31132263 |
| SRA |
SRX17777253 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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