title: GSM4808287: J16_Bulk_P80; Mus musculus; RNA-Seq
library_construction_protocol: Cells were captured via FACS from bulk tumor, TAVs, and TDECs and total RNA was isolated using Trizol (Thermoscientific, Cat. No. 15596018) and the RNeasy Micro Kit (Qiagen, Cat. No. 74004). RNA quality was confirmed on a 12-Capillary Fragment Analyzer using the High Sensitivity RNA Analysis Kit (Agilent formerly AATI, Cat. No. DNF-472-0500). Approximately 5 ng of total RNA was used for cDNA library construction using the Trio RNA-Seq System (0507-96, NuGEN). 8-bp single index libraries were quality controlled using the Standard Sensitivity NGS Fragment Analysis Kit (Agilent formerly AATI, Cat. No. DNF-473-0500,).  Samples were quantified with Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher, Cat. No. Q33120,) and equal concentrations (2 nM) of libraries were pooled, diluted and denatured according to the manufacture's protocol. Libraries were subjected to paired-end (2x75) sequencing of approximately 40 million reads per sample using the High Output v2 kit (Illumina, Cat. No. FC-404-2002) on a NextSeq500. Ultra-Low Input Bulk RNA-Seq of Murine and Human PDX Tumor-Derived Endothelial Subpopulations: Rare, tumor-derived endothelial cells—TDLECs and TDBECs—from both our native CRISPR/Cas9 murine model and human PDOX mice, as well as PDOX bulk tumor cells, were isolated by FACS and were collected in 1x PBS (Thermoscientific, Cat. No. 10010023). Total RNA was extracted using Trizol (Thermoscientific, Cat. No. 15596018) and the RNeasy Micro Kit (Cat. No. 74004, QIAGEN). Approximately 250 pg of total RNA was isolated from 2,000 murine or human PDX-derived TDLECs and TDBECs, as well as bulk tumor PDX-derived cells. cDNA was synthesized using the SMART-Seq Ultra Low Input RNA Kit (Takara, Cat. No. 634889). cDNA concentration and quality were determined using the High Sensitivity NGS Fragment Analysis Kit (Agilent, Cat. No. DNF-474-0500) on a 12-Capillary Fragment Analyzer (Advanced Analytical Technologies Inc.). Quantification of cDNA was confirmed using the Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher, Cat. No. Q33120). Sequencing libraries were produced using the Nextera XT DNA Library Kit (Illumina, Cat. No. FC-131-1024) with 50 pg of cDNA input for tagmentation and ligation at ½ the manufacture recommended volumes. The resulting dual indexed Illumina sequencing libraries were validated using the Standard Sensitivity NGS Fragment Analysis Kit (DNF-473-0500, Agilent formerly AATI) and subjected to paired-end (2x75) sequencing at a depth of approximately 40 million reads per sample using a High Output v2 kit, (Illumina, Cat. No. FC-404-2002) on a NextSeq500, according to the manufacture's protocol.