UGDH knockdown in MDA-MB-231

Transcriptome profiling (RNA-Seq) to evaluate the connection the metabolic enzyme UGDH on gene expression. The RNA libraries were prepared and sequenced at University of Houston Seq-N-Edit Core per protocols. Total libraries were prepared with Ovation® Universal Plus mRNA-Seq (NuGen) using 200 ng input RNA. The size selection for libraries were performed using SPRIselect beads (Beckman Coulter) and purity of the libraries were analyzed using the High Sensitivity DNA chip on Bioanalyzer 2100 (Agilent). The prepared libraries pooled and sequenced using NextSeq 500 (Illumina), generating ~15 million 2×76 bp paired-end reads per samples. The RNA-seq raw fastq data were processed with RNA-Seq Express app within the Illumina BaseSpace app suite (www.basespace.illumina.com), which performed alignment with STAR aligner, assignment of aligned reads to genes, and differential gene expression with DESeq2 Analysis was performed comparing controls (shNT) against individual knockdowns (shU1 or shU2). Then, results from both analyses were compared for the intersection to identify commonly altered genes. mRNA profiles of MDA-MB-231 cells carrying either a control shRNA (shNT) or one of two independent shRNAs targeting UGDH (shUGDH-1, shUGDH-2) were generated by deep sequencing, in triplicate, using Illumina NextSeq 500.