RNA-Seq of SU.86.86 transwell coculture with HPaSteC
Total RNA was extracted from RFP+ tumor cells (BxPC-3-RFP and SU.86.86-RFP) after mono-culture, indirect-coculture, or direct-coculture with fibroblasts (HPaSteC) for 24 hrs. For indirect-coculture, tumor cells and fibroblasts were separated by 0.4um cell culture insert. Fluorescence activated cell sorting was performed to collect RFP+ tumor cells after 24 hrs of mono- or co-culture conditions. Total RNA was subjected to cDNA synthesis and NGS library construction using the Universal Plus mRNA-Seq (Tecan Genomics, San Carlos, CA). The quality and average length of sequence library for each sample was assessed using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and either the DNA 1000 kit. The indexed samples were pooled equimolarly and sequenced on Illumina NovaSeq 6000 (150 base, paired-end reads) (Illumina, San Diego, CA, USA).