Supplementary Figure 2: Description of multiplexed lentiviral vectors, tumor initiation, and Tuba-seq pipeline to quantify tumor size distributions in vivo. | Nature Genetics

Supplementary Figure 2: Description of multiplexed lentiviral vectors, tumor initiation, and Tuba-seq pipeline to quantify tumor size distributions in vivo.

From: Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice

Supplementary Figure 2

a. Lenti-sgTS-Pool/Cre contains four vectors with inert sgRNAs and eleven vectors with tumor suppressor gene targeting sgRNAs. Each sgRNA vector contains a unique sgID and a random barcode. NT = Non-Targeting. b. Schematic of the sgID-barcode region of the vectors in Lenti-sgTS-Pool/Cre. Lenti-sgTS-Pool/Cre contains vectors with fifteen different 8-nucleotide unique identifiers (sgIDs) which link a given sgID-barcode read to a specific sgRNA. These vectors also contain a 15-nucleotide random barcode element. This double barcode system allows identification of individual tumors, as well as the sgRNA in the vector that initiates each tumor. c. Transduction of lung epithelial cells with the barcoded Lenti-sgTS-Pool/Cre pool initiates lung tumors in genetically engineered mouse models with (1) a Cre-regulated oncogenic KrasG12D (KrasLSL-G12D/+) allele, (2) a Cre reporter allele (Rosa26LSL-Tomato), (3) a Cre-regulated Cas9 allele (H11LSL-Cas9), as well as (4) homozygous floxed alleles of either p53 or Lkb1. Lentiviral vectors stably integrate into the genome of the transduced cell. Tumors were initiated in KT;Cas9, KPT;Cas9, and KLT;Cas9 mice to generate 31 different genotypes of lung tumors. Mice were analyzed after 15 weeks of tumor growth. Genomic DNA was extracted from whole lungs, after the addition of barcoded ā€œbenchmarkā€ cell lines, the sgID-barcode region was PCR amplified, deep-sequenced, and analyzed to determine the relative expansion of each uniquely barcoded tumor using the Tuba-seq pipeline.

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