Quantitative RT-PCR and RNA-seq Analysis

Cells were reverse transfected, and total RNA was isolated and cDNA synthesized as previously described (13 (link)–15 (link)). Quantitative Real-Time PCR (qRT-PCR) was performed using PowerUp SYBR Green Master Mix (A25778; Thermo Fisher Scientific) on an ABI StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA) or a CFX384 (Bio-Rad, Hercules, CA). Fold-change gene expression was calculated by the ^ΔΔCT method and normalized to NACA mRNA levels. Samples were generated from three independent experiments unless stated otherwise. For paired-end RNA sequencing (RNA-seq), RNA was extracted (Maxwell 16 LEV simplyRNA purification kit, Promega, Madison, WI), and libraries were prepared and sequenced using standard protocols and a Nextseq 2000 instrument (Illumina, San Diego, CA). The CCR Collaborative Bioinformatics Resource (CCBR) RNA-seq pipeline was used for data analysis https://bioinformatics.ccr.cancer.gov/ccbr/pipelines-software/ccbr-pipeliner/ (see Supplemental Materials and Methods for additional details).