design: mRNA-seq of H8N8 derived mammary tumors, untreated (Ctr.) or upon 11 Gy total dose (Rad.)
library_construction_protocol: H8N8-derived mammary tumors were dissected from WAP-T mice and snap-frozen at liquid nitrogen.  RNA  from  WAP-T  NP8  mammary carcinomas were extracted using a  Trizol/Chloroform protocol according to manufacturer's instructions. For this purpose, shock-frozen mammary tumors from female mice were used, which were stored at  -80  °C. To extract the RNA, approximately 40 mg of frozen tissue was cut for each condition on a dry ice-cooled block with a scalpel. Experiments were conducted with two untreated tumor tissues and four irradiated tumors. All utensils used for the preparation were disinfected with 70 % DEPC-EtOH. Tissues were collected in separate tissue-homogenization tubes filled with 1,4 mm ceramic beads. All the following steps were carried out on ice. 1 ml (EXTRAzol) reagent was added, and the tissues were dissociated in the tissue lyser for three cycles 20 s at 50 Hz and  30  s  break on ice.  After centrifugation  (12.000  x  g,  1  min,  4  °C),  the supernatant was transferred to new tubes and the samples were incubated for  5  min at  RT  to dissociate the nucleotide complexes. For phase separation, 200 μl chloroform was added and the tubes were vortexed for 15 s. The lysate was incubated at RT for 10 min, followed by centrifugation (12.000 x g, 10 min, 4°C). The resulting upper aqueous phase containing the RNAs was transferred to a new 1,5 ml tube and the second round of chloroform extraction was performed, adding again 200 μl chloroform, incubating 5min at RT, centrifuging (12.000 x g, 5 min, 4 °C) and transferring the upper phase to a new 1,5 ml tube. The fresh tubes with 500 μl isopropanol and the upper phases were strongly mixed and incubated at  -80  °C  for at least  1h to precipitate the  RNA. After centrifugation (12.000 x g, 10 min, 4 °C), the supernatant was discarded and the RNA pellet was washed with 80 % ice-cold EtOH (centrifugation 12.000 x g, 10 min, 4°C) and was centrifuged again (8000 x g, 1 min, 4 °C) before air drying to remove excess EtOH. The RNA pellet was resuspended in  50  μl  DEPC-H2O  and  RNA  concentration was measured with DeNovix Spectrophotometer. To improve the quality of the mentioned RNA, a second cycle of RNA isolation was performed, following all the above-mentioned steps for EXTRAzol/Chloroform extraction. For sequencing, mRNA libraries were generated using the NEXTflex Rapid Directional RNA-Seq Library Prep Kit (Bioo Scientific, Austin, TX, US) and sequenced in a HiSeq 4000 automatic sequencer (Illumina, San Diego, CA, US). Total RNA was converted into a library of template molecules suitable for Illumina sequencing using TruSeq RNA Sample Prep kit v2 (Illumina) according to the manufacturer´s instructions adapted for half-volume reactions. Library quality was assessed using an Agilent Bioanalyzer 2100.