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Sample GSM7639395 Query DataSets for GSM7639395
Status Public on Jul 30, 2023
Title KSQ-IVP-0059_GP4_Tumor_CD45_MS4_S1
Sample type SRA
 
Source name PMEL
Organism Mus musculus
Characteristics cell type: PMEL
time: Day 14
library: lib31
sampletype: endpoint
genotype: Cas9 Over-Expression
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).
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 200L 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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
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
 
Submission date Jul 18, 2023
Last update date Jul 30, 2023
Contact name Micah Benson
E-mail(s) mbenson@ksqtx.com
Organization name KSQ Therapeutics
Street address 4 Maguire Rd
City Lexington
State/province MA
ZIP/Postal code 02421
Country USA
 
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
Relations
BioSample SAMN36534104
SRA SRX21065611

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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