2025-03-31
ENA first public
ERC000011
ENA-CHECKLIST
University of Chicago
INSDC center name
public
INSDC status
ERS19897229
SRA accession
E-MTAB-13870_2:Control
Submitter Id
ArrayExpress
broker name
B16
cell_line
not collected
collection date
house mouse
common name
Protocols: To identify tumor cell-intrinsic genes essential for antigen-specific CD8+ T cell killing, we utilized the B16.SIY melanoma cell line which expressed the model antigen SIY (SIYRYYGL) co-expressed with DsRed, that can be recognized by 2C CD8+ TCR transgenic (Tg) T cells.  B16.SIY cells were transduced and selected to stably expressed a Cas9 gene, and then transduced with a genome-scale CRISPR gRNA library.  The library consists of 87,897 sgRNA sequences targeting 19,150 mouse protein-coding genes.  Transduced cells were cultured for 8 days to provide time for gene disruption to occur, then were co-cultured with pre-activated 2C CD8+ T cells at a 2:1 Effector:Target ratio for 16 hours, which we had previously shown results in around 99% killing of WT B16.SIY cells 27.  By performing deep sequencing, we examined the sgRNA library representation in tumor cells with or without T cell co-incubation by MAGeCK analysis Transduced cells were cultured for 8 days to provide time for gene disruption to occur, then were co-cultured with pre-activated 2C CD8+ T cells at a 2:1 Effector:Target ratio for 16 hours, which we had previously shown results in around 99% killing of WT B16.SIY cells The genomic DNA was extracted by MasterPure Complete DNA and RNA Purification Kit Cat No. MC85200 from LGC Biosearch Technologies two-step PCR amplification were performed on gDNA using  Q5® High-Fidelity DNA Polymerases (Neb). The first PCR step (PCR1) included amplification of the region containing sgRNA cassette using gRNA F1 (CTTGAAAGTATTTCGATTTC) and gRNA R (ACTCGGTGCCACTTTTTCAAGT) primers, and the second step PCR (PCR2) included amplification using uniquely Illumina barcoded adaptor-containing primers to allow multiplexing of samples in a single MiSeq.
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
Mus musculus
scientific_name
C57BL/6
strain
Control
title