RNA-sequencing was performed by Fox chase Cancer Center genomics facility (333 Cottman Ave, Philadelphia, PA 19111) and Genewiz (115 Corporate Boulevard, South Plainfield, NJ 07080). HT29 cells were treated with or without 1 μM PG3-Oc in triplicate for 24 h. HCT116 and HCT116 p53−/− were treated with 50 μM 5-FU for 24 h. Total RNA was isolated using RNeasy Mini kit (Qiagen). RNA concentration and quality was analyzed using a NanoDrop 2000. RNA integrity of each sample was analyzed on a Bioanalyzer (Agilent).

Reagents: Truseq stranded mRNA library kit, Hiseq rapid SRcluster kit, HiSeq rapid SBS kit (Illumina,CA). Equipment: HiSeq2500 sequencer (Illumina, CA).

Stranded mRNA-seq library: 1000ng total RNAs from each sample were used to make library according to the product guide. In short, mRNAs were enriched twice via poly-T based RNA purification beads, and subjected to fragmentation at 94° for 8 min via divalent cation method. The first strand cDNA was synthesized by Superscript II and random primers at 42° for 15 mins, followed by second strand synthesis at 16° for 1 h. During second strand synthesis, the dUTP was used to replace dTTP, thereby the second strand was quenched during amplification. A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments at 37° for 30 min. Adapters with illuminaP5, P7 sequences as well as indices were ligated to the cDNA fragment at 30° for 10 min. After Ampure bead (BD) purification, a 15-cycle PCR reaction was used to enrich the fragments. PCR was set at 98° for 10 sec, 60° for 30 sec and extended at 72° for 30 sec.  Libraries were again purified using AmPure beads, had a quality check on bioanalyzer (Agilent) and quantified with Qubit (Invitrogen). Sample libraries were subsequently pooled and loaded to the sequencer. Single end reads at 100 bp were generated for the bioinformatic analysis.

Bioinformatics analysis: Pathway and network analysis (cut-off is 2-fold and above) by Ingenuity Pathway Analysis (IPA; Qiagene) was performed to identify key biological processes, canonical pathways, upstream transcriptional regulators and gene networks. Gene Set Enrichment Analysis was performed by ranking genes first by highest to lowest log 2-fold change. The ranked gene list was then queried using GSEA software to known Molecular Signature Database (MsigDB). Known pathways from curated databases and published studies that matched our gene signature were then reported in the analysis.

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