|Public on Feb 08, 2017
|Purified tumor-associated CD31+ endothelial cells
|tumor cell line: E0771
end timepoint: Tumor size reach 500mm3.
mouse stain: C57BL/6J
genetic background: CD8KO
|0.5 million E0771 tumor cells were injected into the mammary fat pad of C57BL/6J mice with different immunodeficient backgrounds. Tumor growth was monitored every two days, and tumors were resected when tumor volume reached 500mm3.
|The tumors were dissociated into single cell suspensions using the mouse Tumor Dissociation Kit, and endothelial cells were enriched with CD31 microbeads (both from Miltenyi). The isolated cells were subjected to flow cytometry activated cell sorting to further purify tumor-associated CD31+ endothelial cells. The cells were sorted directly into TRIzol LS (Thermo Fisher), and RNA was extracted as per manufacturer's instructions. Double-strand cDNA (dsDNA) were synthesized using Multiple Annealing based RNA-seq approach (MATQ). Illumina Nextera XT DNA Sample Prep Kit was used with 1 ng of dsDNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using Nextera Illumina protocols
|Illumina NextSeq 500
|CD4+ T cell competent background
|Illumina Nextseq 500 automatically used bcl2fastq2 (version 2.17) for basecalling. FASTQ files were downloaded from BaseSpace.
RNA-seq NGS reads were mapped using STAR RNA-seq aligner (version 2.4.1d) and quantified using RSEM (version 1.2.28).
Average insert sizes were calculated for each sample. Consistent with Bioanalyzer electrophoresis plot, one sample (CD4KO_9768) was shown to be an outlier and was removed before proceeding to downstream analysis. An outlier is defined as a number that is more than 1.5 times the inter-quartile range away from either the lower or upper quartiles. Speciﬁcally, if a number is less than Q1 - 1.5×IQR or greater than Q3 + 1.5×IQR, then it is an outlier.
DEseq2 R package was used to normalize the gene expression matrix.
Supplementary_files_format_and_content: 20 RSEM read quantification files for 20 samples (containing Transcript Per Million (TPM) and Fragments Per Kilobase of transcript per Million (FPKM) information); VN.Rdata file contains the sample annotation table and gene expression matrix tables with mouse gene symbols or ortholog human gene symbols as row names.
|Nov 10, 2016
|Last update date
|May 15, 2019
|Memorial Sloan Kettering Cancer Center
|Cancer Biology & Genetics Program
|Mortimer B. Zuckerman Research Center, 417 East 68th Street
|New York City
|RNA-seq of Tumor-associated Endothelial Cells from Different Immunodeficient Backgrounds