GSM4876796: Donor 1 (A5); Homo sapiens; RNA-Seq
After 16hrs, cultured cells were harvested and subjected to a Dead Cell Removal kit (Miltenyi Biotec) prior to staining for flow cytometric analysis and isolating RNA using RNeasy Plus Mini kits (Qiagen, Hilden, Germany).  RNA samples were submitted to the Dana-Farber Cancer Institute Molecular Biology Core Facilities (Boston, MA) for RNA-seq analysis. For library preparation, libraries were prepared using Roche Kapa mRNA HyperPrep sample preparation kits from 100ng of purified total RNA according to the manufacturer's protocol.  The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to manufacturer's protocols. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with single-end 75bp reads.  For RNAseq analysis, sequenced reads were aligned to the UCSC hg19 reference genome assembly and gene counts were quantified using STAR (v2.5.1b) (55). Differential gene expression testing was performed by DESeq2 (v1.10.1) (56) and normalized read counts (FPKM) were calculated using cufflinks (v2.2.1) (57). RNA-seq analysis was performed using the VIPER snakemake pipeline (58). Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with single-end 75bp reads.  For RNAseq analysis, sequenced reads were aligned to the UCSC hg19 reference genome assembly and gene counts were quantified using STAR (v2.5.1b) (55). Differential gene expression testing was performed by DESeq2 (v1.10.1) (56) and normalized read counts (FPKM) were calculated using cufflinks (v2.2.1) (57). RNA-seq analysis was performed using the VIPER snakemake pipeline (58).