NextSeq 500 sequencing; RNA-seq of human colorectal cancer cell line COLO320DM treated with the secretome of irradiated or control cancer-associated fibroblasts for 6 or 48 hours
RNA-seq of human colorectal cancer cell line COLO320DM treated with the secretome of irradiated or control cancer-associated fibroblasts for 6 or 48 hours
COLO320DM cells were seeded and 24 hours later 1.8Gy irradiated. Subsequently, 24h later COLO320DM cells were treated with the secretome of 10x 1.8Gy irradiated CAFs or sham treated CAFs in 0.5% of serum. RNA was isolated 6 hours or 48 hours later. After trypsinization of COLO320DM, a solution of 5 x 106 cells was centrifuged (210 g, 5 min, 4°C) and cell pellets were washed twice with PBS. The supernatant was discarded and the cell pellet was stored at -80°C.  RNA was isolated with the miRNeasy kit (Qiagen, Venlo, The Netherlands) Libraries for mRNA sequencing were prepared using the TruSeq stranded mRNA sample prep kit (Illumina). The starting material (100 ng) of total RNA was mRNA enriched using the oligodT bead system (Illumina). The isolated mRNA was subsequently fragmented using enzymatic fragmentation. Then first strand synthesis and second strand synthesis were performed and the double stranded cDNA was purified (Agencourt AMPure XP, Beckman Coulter). The cDNA was end repaired, 3' adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (Agencourt AMPure XP). The mRNA stranded libraries were pre-amplified with PCR and purified (Agencourt AMPure XP). The libraries size distribution was validated and quality inspected on the 2100 Bioanalyzer
Experimental Factor: secretome of irradiated cancer-associated fibroblasts
stimulus
Experimental Factor: 6
time