Illumina HiSeq 2500 sequencing; RNA-Seq of native and FusionRed-transfected canine tumor cell lines after 3 hours C-CPE treatment
Canine tumor cell lines TihoDProCarc0840 (0840) and TihoDProAdCarc0846 (0846) were derived from canine prostate carcinomas previously described by Reimann-Berg et. al (2011) and Packeiser et al. (2020). Both cell lines express CLDN3, -4 and -7 (Hammer et. al. 2016).Transfected cell lines 0840-FusionRed and 0846-FusionRed (transfected with pFusionRed-C (Cat.#FP411, Evrogen)) were generated and characterized. C-CPE was prepared as described previously by Becker et. al. 2019. Native and transfected cell lines were seeded in triplicate with a density of 5*105 cells in 6-well plates 48 hours to reach monolayer. C-CPE with a concentration of 20 µg/ml was added and the cells incubated for 3 hours to allow binding of C-CPE to CLDNs. In the next step, culture medium was removed, and cells were washed with 5ml phosphate buffered saline (PBS). TrypLETM Express (Gibco by Life technologiesTM, Darmstadt, Germany) was used for detaching cells, centrifugation at 1000 rpm for 10 min followed for pelleting. Pellets were stored at -80 °C and followed by RNA-isolation for transcriptome analysis.  The cells were cultivated separately in 25 cm2 cell culture flasks in medium 199 (Gibco by Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal calf serum (FCS Superior, Biochrom GmbH, Berlin, Germany) and 2 % penicillin/streptomycin (Biochrom GmbH, Berlin, Germany), and incubated in a humidified incubator maintained at 37 ˚C with 5 % CO2. Cultivation medium was replaced twice per week.   Total RNA was isolated from transfected and native prostate tumor cell lines with C-CPE treatment and without C-CPE treatment using the RNeasy®Mini Kit RNA Purification (Qiagen, Hilden, Germany) according to the manufacturer´s instruction. RNA isolation was performed three times per cell line. On-column DNase digestion was carried out with RNase-Free DNase Set (Qiagen Hilden, Germany) to avoid genomic DNA contamination.The RNA quality was assessed using an Agilent RNA 6000 Nano kit and 2100 Bioanalyzer (Agilent).  Samples with RNA integrity number (RIN) > 8 were used for the DNA library preparation using a TruSeq Stranded mRNA Sample Preparation kit according to the manufacture's protocol (Illumina). Briefly, 1 µg of total RNA was used as input for an mRNA enrichment using poly-T oligo coated magnetic beads and chemically fragmented under elevated temperature. The fragmented RNA was then reverse-transcribed into the first- and second-strand cDNA using random hexamers and Superscript II reverse transcriptase. Double stranded cDNA fragments were ligated with TruSeq RNA adapters with a unique DNA sequencing index and PCR-amplified. The DNA libraries were quality-controlled using an Agilent Technologies 2100 Bioanalyzer and Agilent DNA-1000 Chip kit.