RNA-Seq of HPaSteC direct coculture with PANC-1
Total RNA was extracted from RFP+ fibroblasts (HPaSteC-RFP) after mono-culture, indirect-coculture, or direct-coculture with tumor cells (BxPC-3, SU.86.86, MIA PaCa-2, and PANC1) 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+ fibroblasts 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).