2.9. RNA Isolation and RNA-Sequencing

Total RNA was isolated from snap-frozen tumor tissue specimens using TRIzol-chloroform extraction. RNA was processed for sequencing using the RNeasy Mini Kit (Qiagen, Germantown, MA, USA). Purified RNA concentration and quality was determined by ND-1000 spectrophotometer using NanoDrop 3.0.1 software (Coleman Technologies Inc., Wilmington, DE, USA). RNA samples (200 ng) were analyzed at the UAB Heflin Center Genomics Core (Birmingham, AL, USA). The RNA-seq libraries were prepared on the NextSeq500 Illumina Sequencing Platform (Illumina, San Diego, CA, USA) with paired end 75 bp at 40M reads.