Identification of novel DVL3 cancer-associated target genes [RNA-seq]

The goal of this study is to identify novel target genes of DVL3 in two breast cancer cell lines Methods:Total RNA was isolated using the Aurum™ Total RNA Mini Kit (Bio Rad) and library preparation and sequencing were performed at Center for Biotechnology & Genomics of Texas Tech University. RNA quality was determined using RNA Screen Tape (Agilent). Ribosomal RNA depletion was achieved using NEB Next rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310X). RNA fragmentation, double stranded cDNA and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep according to the manufacturer’s protocol (NEB # E7760L). PCR enriched libraries were quantified by Qubit and equimolar indexed libraries (different samples had different indexes for multiplexing) were pooled. Pooled libraries were quantitatively checked using the Agilent Tapestation 2200 and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 SP flow cell, paired-end reads (2 × 50 bp). We compared non-target control (WT) vs. DVL3 KD (shB), and empty vector (EV) vs. DVL3-WT in 3 cell lines MDA-MB-468 and MCF7