Computational protocol: Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors

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Protocol publication

[…] E0771 cells were purchased from CH3 Biosystems (Amherst, New York), and cultures were maintained with 10% FBS (Atlanta Biologicals), 1% penicillin-streptomycin (ThermoFisher), and 10 mM HEPES (ThermoFisher) in DMEM. Total RNA was extracted from E0771 cultures using the Qiagen RNeasy Mini Kit according to the manufacturer's protocol. RNA-sequencing libraries were prepared with 3 μg total RNA input using the Illumina TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's protocol. Sequencing was performed on the Illumina NextSeq500 with the help of the Cedars-Sinai Medical Center Genomics Core generating approximately 60 M and 23 M reads. Reads were aligned to the mm9 version of the mouse reference genome using the STAR aligner. The GATK pipeline was used to call variants from the aligned data to generate a VCF file, which was subsequently annotated using SnpEff and filtered for Trp53 using SnpSift. […]

Pipeline specifications

Software tools STAR, GATK, SnpEff, SnpSift
Application RNA-seq analysis
Diseases Breast Neoplasms, Neoplasms