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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 30, 2023 |
| Title |
KSQ-IVP-0059_Input3_lib30_S7 |
| Sample type |
SRA |
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| Source name |
PMEL
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| Organism |
Mus musculus |
| Characteristics |
cell type: PMEL
time: Day 0
library: lib30
sampletype: input
genotype: Cas9 Over-Expression
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| Treatment protocol |
The ‘Lib30’ sgRNA library targets 369 genes involved in T cell function with 10sgRNAs per gene and 3089 sgRNAs total, including controls, was cloned into pKSQ044. Lib31 sgRNA library targets 1004 predicted CSR genes with 8 sgRNAs/gene and 11,148 sgRNAs total, including controls, was also cloned into pKSQ044 (see Supplementary Methods for more information on library design and lentivirus production).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cas9-Tg x TCR-Tg OT1 or PMEL CD8 T cells were isolated from freshly harvested mouse spleens and dissociated using a GentleMACS (Miltenyi), and with CD8 T cells purified by negative selection (EasySep Mouse CD8+ T cell isolation kit). CD8s were then placed in T225 flasks at a concentration of 1M CD8s/ml and activated with CD3/CD28 Dynabeads and 2ng/ml mouse rIL-2. The following day, T cells were transduced wither either lentivirus (OT1 B16-Ova screen) or retrovirus (PMEL / MC38-gp100 screen). Dynabeads were removed the following day, with cells resuspended on cRPMI + 32ng/ml IL-2. On Day 4, transduced T cells were harvested and selected by positive selection using either Thy1.1 for OT1s or hCD2 for PMELs. 5x106 Thy1.1+ Cas9-Tg x OT1 CD8 T cells or 7x106 hCD2+ Cas9-Tg x PMEL CD8 T cells were injected into tumor-bearing mice i.v. via tail vein in 200L PBS, with 10x106 CD8+ T cells saved to determine the sgRNA distribution of the input population of T cells. Group sizes were 7-8 mice per library. At the indicated time (eg 11-21 days) following adoptive transfer of cells, mice were euthanized and blood was harvested in EDTA tubes, and stored at -80C. Tumors, spleens, tumor draining and non-draining lymph nodes were harvested and processed further for extraction of CD8 T cells using CD8a Microbeads (Miltenyi, cat# 130-049-401) from the spleen and CD45 Microbeads (Miltenyi, cat# 130-052-301) from the tumor. Tumors were digested using the Miltenyi Tumor Dissociation Kit (Cat# 130-096-730) according to the manufacturer’s instructions.
Genomic DNA was isolated using the Qiamp Blood Midi and Maxi kids, according to the manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Read and UMI counts were tabulated using linux command line programs awk, sed, sort, uniq, zcat and paste
Sequencing-errors in guide and UMI sequences were suppressed by aggregating guides and UMIs with an edit-distance of one
UMIs that did not match the synthesized degenerate UMI-pattern were removed
UMIs potentially representing index-swapping between samples on the same sequencing run were removed
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Library strategy: Pooled CRISPR Screen
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| Submission date |
Jul 18, 2023 |
| Last update date |
Jul 30, 2023 |
| Contact name |
Micah Benson |
| E-mail(s) |
mbenson@ksqtx.com
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| Organization name |
KSQ Therapeutics
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| Street address |
4 Maguire Rd
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| City |
Lexington |
| State/province |
MA |
| ZIP/Postal code |
02421 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE237691 |
Rational design of a SOCS1-edited tumor infiltrating lymphocyte therapy for solid tumors using CRISPR/Cas9 screens [IVP59_mc38gp100] |
| GSE237695 |
Rational design of a SOCS1-edited tumor infiltrating lymphocyte therapy for solid tumors using CRISPR/Cas9 screens |
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| Relations |
| BioSample |
SAMN36534082 |
| SRA |
SRX21065617 |
| 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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