Nebnext unique dual index primers

Total RNA was extracted from cells treated as outlined above. The NEBNext^® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) was used as per manufacturer protocol to isolate mRNA for input into the NEBNext^® Ultra^™ II RNA Library Prep Kit (New England Biolabs) using NEBNext Unique Dual Index Primers (New England Biolabs). Library preparation was conducted as per manufacturer protocol. Libraries were quality checked using Agilent D1000 TapeStation kit and Qubit Flourometer. FLAG-FOXM1-hTERT RPE1 experimental samples were sequenced on an Illumina NextSeq (Shilatifard lab) to generate 2×75 bp paired-end reads and FOXM1 and CENP-F siRNA experimental samples were sequenced on an Illumina HiSeq (Admera Health) to generate 2×150 bp paired-end reads. We obtained 40M reads per sample. Data analysis including read demultiplexing, alignment and differential expression were done using the Ceto pipeline (https://github.com/ebartom/NGSbartom) with standard parameters. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes was performed using the Molecular Signature DataBase from the Broad Institute[52 (link), 53 ]. TCGA Pancancer Atlas datasets were downloaded from cBioPortal (https://www.cbioportal.org) as mRNA expression z-scores relative to normal samples (log RNA Seq V2 RSEM). Downstream analyses were conducted using R.